AU2017271189B2 - Cytosol-penetrating antibody and use thereof - Google Patents

Abstract

The present invention relates to a cytosol-penetrating antibody and a use thereof, and specifically, to a light chain variable region and/or heavy chain variable region positioned in a cytosol, a cytosol-penetrating antibody containing same, a method for producing same, and a use thereof, wherein the light chain variable region and/or heavy chain variable region exhibit notably enhanced endosomal escape efficiency as a result of an investigation of a structural mechanism for inducing endosomal escape which is the escape from an endosome to a cytosol after cellular internalization through a membrane protein of a cell membrane of a living cell.

Description

CYTOSOL-PENETRATING ANTIBODY AND USE THEREOF TECHNICAL FIELD

The present disclosure relates to a cytosol-penetrating

antibody and the use thereof, and more particularly to an

endosomal escape motif which can increase the endosomal

escape efficacy of a cytosol-penetrating antibody

(Cytotransmab) so as to have a significantly improved ability

to escape from endosomes into the cytosol after cellular

internalization into living cells through a cell membrane

protein, a light-chain variable region and/or heavy-chain

variable region comprising the same, a cytosol-penetrating

antibody comprising the same, a method for producing the same,

and the use thereof.

BACKGROUND ART

General antibodies and macromolecular bio-drugs have

limitations in that they cannot pass the hydrophobic cell

membrane, and thus cannot bind to and inhibit various

disease-related substances. In addition, conventional

antibodies cannot directly penetrate living cells due to

their large size and hydrophilic nature.

Thus, most conventional antibodies specifically target

extracellularly secreted proteins or cell membrane proteins.

Further, generally, commercial antibodies binding

specifically to intracellular substances which are used in

experiments for studies on mechanisms such as the growth,

specific inhibition, etc. of cells, cannot be used directly

to treat living cells, and in order for these antibodies to

bind to intracellular substances, a pretreatment process for

forming pores in the cell membrane by a cell membrane

permeabilization process using the amphipathic glycoside

saponin is necessarily required.

A number of therapeutic antibodies that target cell

membrane proteins or extracellularly secreted proteins due to

their property of binding to target proteins with high

specificity and high affinity have been developed. Antibodies

that target cell membrane proteins can bind to cell membrane

proteins, and then enter the cells via an endosomal pathway

through a receptor-mediated endocytosis process.

This process includes various pathways after the early

endosome stage. That is, 1) most antibodies can be

transported from early endosomes to late endosomes and

lysosomes, and can be completely degraded by proteases under

acidic conditions; and 2) some antibodies can bind to FcRn

(neonatal Fc receptor) in early endosomes under acidic

conditions and come out of the cells through the recycling

endosome pathway.

Thus, most antibodies bind strongly to the target

membrane proteins and are mostly degraded through the

lysosomal pathway. In the endosomal pathway, endosomes are

matured while the inside thereof is gradually acidified by

proton pumps. It is known that the pH of early endosomes is

about 5.5-6.5, the pH of late endosomes is about 4.5-5.5, and

the pH of lysosomes is about pH 3.5-4.5 (Quadir MA et al.,

2014; Li S et al., 2014). Many proteinases in endosomes are

activated, and endocytosed proteins are degraded in endosomes.

Consequently, when antibodies move through the endosomal

pathway after receptor-mediated endocytosis, they should be

separated from the target membrane proteins and form pores in

endosomes in order to escape from early or late endosomes

into the cytosol before trafficking to lysosomes.

Among naturally occurring intracellular substances,

viruses and toxins are known to actively penetrate living

cells through endocytosis. "Endosomal escape", a process of

escaping from endosomes into the cytosol, is essential so

that a substance that penetrated into cells by endocytosis

exhibits activity in the cytosol.

Although the endosomal escape mechanism has not yet been

clearly found, three hypotheses for the endosomal escape are

known to date.

The first hypothesis is a mechanism by which a pore is

formed in the endosomal membrane. In this hypothesis, substances such as cationic amphiphilic peptides in the endosomal membrane bind to a negatively charged cellular lipid bilayer to cause internal stress or inner membrane contraction to thereby form a barrel-stave pore or a toroidal channel (Jenssen et al., 2006), which is called a pore formation mechanism.

The second hypothesis is a mechanism by which the

endosome bursts as a consequence of the proton-sponge effect.

In this hypothesis, due to the high buffering effect of a

substance having a protonated amino group, the osmotic

pressure of the endosome can be increased so that the

endosomal membrane can be degraded (Lin and Engbersen, 2008).

In the third hypothesis, a specific motif, which

maintains a hydrophilic coil shape in a neutral environment

but is changed into a hydrophobic helical structure in an

acidic environment such as endosome, is fused to the

endosomal membrane so that viruses and toxins including a

motiff escape from the endosome, which is called a lipid

membrane fusion mechanism. These three hypothese has been

proposed as endosome escape mechanisms after endocytosis of a

viral protein and a toxic protein derived from

plants/bacteria, but such endosome escape mechanism in

antibodies has not been specifically identified yet.

The common phenomenon observed in the above-described

endosomal escape mechanism is that endosomal escape occurs under acidic pH conditions which are endosomal and lysosomal environments. Proteins whose function changes depending on pH have the property of changing their structure depending on pH.

Negatively charged amino acids (aspartic acid (D) and

glutamic acid (E)) and hydrophobic amino acids (methionine

(M), leucine (L), and isoleucine (I)) do not interact under

neutral pH conditions. However, as pH decreases, the

carboxylic acids (COO-) in the side chains of the negatively

charged amino acids become hydrophobic by protonation (Korte

et al., 1992), and then can hydrophobically interact with the

surrounding hydrophobic amino acids. As a result, the

distance between the two amino acids becomes closer, and the

overall structure and function of the protein change. The

phenomenon that causes this change is known as the Tanford

transition (Qin et al., 1998).

As one example, nitrophorin 4, a nitrogen transporting

enzyme, has an open structure under neutral pH conditions.

However, as the pH decreases from neutral pH (pH 7.4) to

weakly acidic pH (pH 6.0), the structure of nitrophorin 4

changes to a closed structure by the hydrophobic interaction

of aspartic acid and leucine, and thus nitrophorin 4

functions to transport nitrogen (Di Russo et al., 2012).

However, this pH-dependent structural change has not yet

been found in antibodies. In particular, this change has not

yet been observed in antibodies that undergo endocytosis.

As one example, an antibody engineering improvement

technology for inducing pH-dependent antigen binding among

conventional antibody technologies is a method of screening

pH-dependent antigen-binding antibodies from libraries

introduced either with histidine (H) of CDRs (complementary

determining regions) or with random mutations including

histidine (Bonvin P et al., 2015). However, the two methods

all cause no structural change, and have a limitation in that

library-based screening should be performed in order to

induce pH-dependent antigen binding.

In order to increase the effect of a substance that

exhibits its activity in the cytosol, the amount of the

substance located in the cytosol should ultimately increase.

Hence, studies have been conducted to increase the endosomal

escape ability. Such studies have been conducted mainly on

cell-penetrating peptides (CPPs). Although it has been

reported that some cell-penetrating peptides localize in the

cytosol through an endosomal escape pathway, there is no

detailed study on an exact endosomal escape mechanism, and it

is known that only about 0.1 to 4% of endocytosed peptides

localize to the cytosol due to very low endosomal escape

efficiency.

Under this technical background, the present inventors

have identified an endosomal escape motif capable of

increasing the endosomal escape efficacy of a cytosol penetrating antibody (Cytotransmab) that penetrates cells and localizes in the cytosol (Choi et al., 2014), and have found that it is possible to develop a light-chain or heavy-chain variable region including an endosomal escape motif having an increased ability to escape from endosomes, and an antibody or an antigen-binding fragment thereof comprising the same.

In addition, the present inventors have found that a

cytosol-penetrating antibody having endosomal escape ability

can be produced by grafting this endosomal escape motif into

other kinds of light-chain or heavy-chain variable regions,

thereby completing the present disclosure.

The information disclosed in the Background Art section

is only for the enhancement of understanding of the

background of the present disclosure, and therefore may not

contain information that forms a prior art that would already

be known to a person of ordinary skill in the art.

DISCLOSURE OF INVENTION TECHNICAL PROBLEM

It is an object of the present disclosure to provide a

cytosol-penetrating antibody having endosome escape ability

or an antigen-binding fragment thereof.

Another object of the present disclosure is to provide a

nucleic acid encoding the antibody or antigen-binding

fragment thereof.

Still another object of the present disclosure is to

provide a vector comprising the above-described nucleic acid,

a cell transformed with the above-described vector, and a

method of producing the above-described antibody or antigen

binding fragment thereof.

Yet another object of the present disclosure is to

provide an antibody-drug conjugate comprising the above

described antibody or antigen-binding fragment thereof.

A further object of the present disclosure is to provide

a composition for delivering an active substance into cytosol,

comprising the above-described cytosol-penetrating antibody

or antigen-binding fragment thereof.

A still further object of the present disclosure is to

provide a method for producing the above-described cytosol

penetrating antibody or antigen-binding fragment thereof.

TECHNICAL SOLUTION

To achieve the above object, the present disclosure

provides a cytosol-penetrating antibody or an antigen-binding

fragment thereof comprising a light-chain variable region

and/or heavy-chain variable region that comprises an endosomal

escape motif in its CDR3, wherein the endosomal escape motif

comprises a sequence represented by the following formula:

X1-X2-X3-Z1 wherein each of Xl, X2 and X3 is selected from the group consisting of tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine (F), wherein the endosomal escape motif comprise one or more tryptophan (W);

Z1 is selected from the group consisting of methionine

(M), isoleucine (I), leucine (L), histidine (H), aspartic

acid (D), and glutamic acid (E);

the 1st amino acid of the light-chain variable region

and/or heavy-chain variable region is aspartic acid (D) or

glutamic acid (E);

the light-chain variable region and/or heavy-chain

variable region comprising Z1 induces a change in properties

of the antibody under endosomal acidic pH conditions; and

the antibody exhibits an ability to escape from

endosomes into the cytosol through the change in properties of

the antibody.

In the cytosol-penetrating antibody or antigen-binding

fragment thereof according to the present disclosure, the

first amino acid of the light-chain variable region and/or

heavy-chain variable region may be aspartic acid (D) or

glutamic acid (E).

The present disclosure also provides a nucleic acid

encoding the above-described cytosol-penetrating antibody or

antigen-binding fragment thereof.

The present disclosure also provides a vector comprising

the above-described nucleic acid.

The present disclosure also provides a cell transformed

with the above-described vector.

The present disclosure also provides a composition for

delivering an active substance into cytosol, comprising the

above-described cytosol-penetrating antibody or antigen

binding fragment thereof.

The present disclosure also provides a method for

producing the above-described cytosol-penetrating antibody or

antigen-binding fragment thereof, the method comprising a step

of grafting the endosomal escape motif X1-X2-X3-Z1 (wherein

X1-X2-X3 is selected from the group consisting of tryptophan

(W), tyrosine (Y), histidine (H), and phenylalanine (F)) into

the CDR3 of a light-chain and/or heavy-chain variable region.

ADVANTAGEOUS EFFECTS

The cytosol-penetrating antibody or antigen-binding

fragment thereof comprising the light-chain variable region

and/or heavy-chain variable region comprising the endosomal

escape motif according to the present disclosure penetrates

living cells and localizes in the cytosol, and ultimately the

antibody or antigen-binding fragment thereof can be penetrate

living cells and localize in the cytosol without having to use

a special external protein delivery system.

The cytosol-penetrating antibody or antigen-binding

fragment thereof according to the present disclosure is a

cytosol-penetrating antibody or antigen-binding fragment

thereof comprising a light-chain variable region or a heavy

chain variable region that easily interacts with and binds to

various human light-chain variable regions or heavy-chain

variable regions (VHs), and has the ability to escape from

endosomes into the cytosol. The antibody or antigen-binding

fragment thereof can penetrate cells and localize in the

cytosol, and does not show non-specific cytotoxicity for

target cells.

Based on the endosomal escape mechanism for the high

efficiency cytosol-penetrating antibody or an antigen-binding

fragment thereof according to the present disclosure, a design

of antibody libraries for improving the endosomal escape

ability and mutants can be performed.

The endosomal escape motif included in the cytosol

penetrating antibody or antigen-binding fragment thereof

according to the present disclosure is introduced into other

antibodies so that it can be expected to impart the endosomal

escape ability.

In addition, the cytosol-penetrating antibody or

antigen-binding fragment thereof according to the present

disclosure can be utilized as a carrier that delivers an

active substance into the cytosol of a living cell, and can also be utilized as a pharmaceutical composition for treatment and prevention of diseases.

Throughout this specification the word "comprise", or

variations such as "comprises" or "comprising", will be

understood to imply the inclusion of a stated element, integer

or step, or group of elements, integers or steps, but not the

exclusion of any other element, integer or step, or group of

elements, integers or steps.

Any discussion of documents, acts, materials, devices,

articles or the like which has been included in the present

specification is not to be taken as an admission that any or

all of these matters form part of the prior art base or were

common general knowledge in the field relevant to the present

disclosure as it existed before the priority date of each of

the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows of a pulse-chase experiment and confocal

microscopy observation performed to observe the transport

- 11A- process and stability of the cytosol-penetrating antibody

(cytotransmab) TMab4 or cell-penetrating peptide TAT

introduced into cells.

FIG. 2a shows the results of confocal microscopy

observation of the cytosol-penetrating ability of the

cytosol-penetrating antibody TMab4 or the cell-penetrating

peptide TAT in the presence or absence of an inhibitor

thereof.

FIG. 2b is a bar graph showing the results of

quantifying the FITC (green fluorescence) fluorescence of the

confocal micrographs shown in FIG. 2a.

FIG. 2c shows the results of observing the cytosolic

localization of the cytosol-penetrating antibody TMab4 or the

cell-penetrating peptide TAT by confocal microscopy using

calcein in the presence or absence of an inhibitor thereof.

FIG. 2d is a bar graph showing the results of

quantifying the calcein fluorescence of the confocal

micrographs shown in FIG. 2c.

FIG. 3a shows the results of Western blot analysis

performed to confirm siRNA (short interfering RNA)-induced

inhibition of heparanase expression.

FIG. 3b shows the results of confocal microscopy

observation of cytosol penetrating antibody/lysosome merging

caused by inhibition of heparanase expression.

FIG. 3c shows the results of confocal microscopy

observation performed to confirm the cytosolic localization

of a cytosol-penetrating antibody, which is caused by

inhibition of heparanase expression.

FIG. 4 is a schematic view showing an overall

trafficking process ranging from cellular internalization of

a cytosol-penetrating antibody to localization of the

antibody in the cytosol.

FIG. 5 shows the results of observing a fluorescence

labeled cytosol-penetrating antibody in Ramos cells by

confocal microscopy in order to examine whether the antibody

can be introduced through the cell membrane depending on pH

or whether the antibody can induce cell membrane permeation

of other substances.

FIG. 6a shows the results of observing Ramos cells by an

optical microscope in order to examine whether a cytosol

penetrating antibody can form pores and take up trypan blue

having no membrane-permeating ability, depending on pH.

FIG. 6b is a graph quantitatively comparing the number

of cells that have taken up trypan blue.

FIG 7a shows the results of optical microscopic

observation performed to confirm whether cell membrane pores

produced by a cytosol-penetrating antibody at pH 5.5 is

temporary and reversible.

FIG. 7b is a graph quantitatively comparing the number

of cells that have taken up trypan blue uptake.

FIG. 8 shows the results of analyzing the cell membrane

binding of a cytosol-penetrating antibody and control

antibody adalimumab by flow cytometry (FACS) at varying pHs.

FIG. 9 shows the results of analyzing the cell membrane

flip-flop inducing abilities of a cytosol-penetrating

antibody and control antibody adalimumab by flow cytometry

(FACS) at varying pHs.

FIG. 10 is a schematic view showing a pore formation

model of a cytosol-penetrating antibody, expected based on

the above-described experiments.

FIG. 11 shows the results of predicting the pH-dependent

structural change of a cytosol-penetrating antibody on the

basis of the WAM modeling structure of the light-chain

variable region of the cytosol-penetrating antibody, and

shows amino acids, which are involved in the structural

change, and amino acids which are exposed by the structural

change.

FIG. 12 is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by

mutants constructed by substituting the 1st amino acid

aspartic acid and 9 5 th amino acid methionine of a light-chain

variable region (VL), which induce a change in properties of a cytosol-penetrating antibody at acidic pH, with alanine, glutamic acid and leucine, respectively.

FIG. 13 is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by

mutants constructed by substituting particular amino acids of

the CDR3 of the light-chain variable region (VL) of a

cytosol-penetrating antibody, which can possibly be involved

in endosomal escape, with alanine.

FIG. 14a shows the results of confocal microscopy

performed to analyze the cytosol-penetrating ability of

mutants constructed by substituting the CDR1 and CDR2 of the

light-chain variable region (VL) of a cytosol-penetrating

antibody, which bind to HSPG receptor and are involved in

cytosol-penetrating ability, with human germline sequences.

FIG. 14b shows a graph quantitatively comparing the

number of cells that have taken up trypan blue depending on

pH by mutants constructed by substituting the CDR1 and CDR2

of the light-chain variable region (VL) of a cytosol

penetrating antibody, which bind to HSPG receptor and are

involved in cytosol-penetrating ability, with human germline

sequences.

FIG. 15a shows the results of 12% SDS-PAGE analysis

under reducing or non-reducing conditions after purification

of cytosol-penetrating antibody mutants expected to have

improved endosomal escape ability.

FIG. 15b shows the results of confocal microscopy

performed to examine whether the cytosol-penetrating ability

of cytosol-penetrating antibody mutants expected to have

improved endosomal escape ability is maintained.

FIG. 16 is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by a

cytosol-penetrating antibody wild-type and cytosol

penetrating antibody mutants expected to have improved

endosomal escape ability.

FIG. 17a shows the results of observing the cytosolic

localization of a cytosol-penetrating antibody wild-type and

cytosol-penetrating antibody mutants expected to have

improved endosomal escape ability, by confocal microscopy

using calcein.

FIG. 17b is a bar graph showing the results of

quantifying the calcein fluorescence of the confocal

micrographs shown in FIG. 17a.

FIG. 18 is a schematic view showing a process in which

GFP fluorescence by enhanced split-GFP complementation is

observed when a cytosol-penetrating antibody wild-type and a

mutant having improved endosomal escape ability localizes in

the cytosol.

FIG. 19 shows the results of 12% SDS-PAGE analysis under

reducing or non-reducing conditions after purification of a

GFP11-SBP2-fused cytosol-penetrating antibody wild-type and a

GFP11-SBP2-fused mutant having improved endosomal escape

ability.

FIG. 20a shows the results of confocal microscopy

performed to examine the GFP fluorescence of a GFP11-SBP2

fused cytosol-penetrating antibody wild-type and a GFP11

SBP2-fused mutant having improved endosomal escape ability by

enhanced split-GFP complementation.

FIG. 20b is a graph showing the results of quantifying

the GFP fluorescence of the confocal micrographs shown in FIG.

20a.

FIG. 21a is a graph showing the results of flow

cytometry (FACS) performed to analyze the cell membrane

binding of mutants obtained by substitution with arginine,

isoleucine and glycine, which are amino acids having

properties opposite to those of tryptophan.

FIG. 21b is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by

mutants obtained by substitution with arginine, isoleucine

and glycine, which are amino acids having properties opposite

to those of tryptophan.

FIG. 21c is a bar graph showing the results of observing

the cytosolic localization of mutants obtained by

substitution with arginine, isoleucine and glycine, which are

amino acids having properties opposite to those of tryptophan by confocal microscopy using calcein and quantifying the calcein fluorescence of the confocal micrographs.

FIG. 22a is a schematic view showing a process of

constructing an intact IgG-format anti-tubulin cytosol

penetrating antibody to be used to examine the activity of

cytosol-penetrating antibody mutants having improved

endosomal escape ability.

FIG. 22b shows the results of 12% SDS-PAGE analysis

under reducing or non-reducing conditions after purification

of an intact IgG-format anti-tubulin cytosol-penetrating

antibody.

FIG. 22c shows the results of confocal microscopy

performed to examine whether an intact IgG-format anti

tubulin cytosol-penetrating antibody would merge with

cytoskeletal tubulin localized in the cytosol.

FIG. 23a is a schematic view showing a process of

constructing an intact IgG-format RAS-targeting cytosol

penetrating antibody to be used to examine the activity of

mutants having improved endosomal escape ability.

FIG. 23b shows the results of 12% SDS-PAGE analysis

under reducing or non-reducing conditions after purification

of intact IgG-format RAS-targeting cytosol-penetrating

antibodies.

FIG. 23c shows the results of enzyme linked

immunosorbent assay performed to measure the affinities of antibodies for GppNHp-bound K-RAS G12D and GDP-bound K-RAS

G12D, which are K-RAS mutants.

FIG. 24 shows the results of confocal microscopy

observation performed to examine whether intact IgG-format

RAS-targeting cytosol-penetrating antibodies would merge with

intracellular H-RAS G12V mutants.

FIG. 25a is a graph showing the results of

quantitatively comparing the number of cells that have taken

up trypan blue depending on pH by mutants constructed by

substituting the 1st amino acid aspartic acid of the light

chain variable region (VL) of a cytosol-penetrating antibody,

which induce a change in properties of the cytosol

penetrating antibody at acidic pH 5.5, with various amino

acids.

FIG. 25b is a graph showing the results of

quantitatively comparing the number of cells that have taken

up trypan blue depending on pH by mutants constructed by

substituting 9 5 th amino acid methionine of the light-chain

variable region (VL) of a cytosol-penetrating antibody, which

induce a change in properties of the cytosol-penetrating

antibody at acidic pH 5.5, with various amino acids.

FIG. 26a shows a graph showing quantitatively comparing

the number of cells that have taken up trypan blue depending

on pH by mutants designed for the purpose of inducing an

additional change in properties in response to pH.

FIG. 26b shows a bar graph showing the results of

observing the cytosolic localization of mutants designed for

the purpose of inducing an additional change in properties in

response to pH by confocal microscopy using calcein and

quantifying the calcein fluorescence of the confocal

micrographs.

FIG. 27 is a graph quantitatively comparing the number

of cells that taken up trypan blue depending on pH by mutants

obtained by changing the amino acid number of the CDR3 of the

light-chain variable region of a cytosol-penetrating antibody.

FIG. 28a shows a process of constructing an intact IgG

format RAS-targeting cytosol-penetrating antibody in which an

improved endosomal escape motif is introduced into the light

chain variable region of a conventional therapeutic antibody.

FIG. 28b shows the results of fluorescence microscopic

observation performed to examine whether the HSPG binding

affinity and cytosol-penetrating ability of an intact IgG

format RAS-targeting cytosol-penetrating antibody in which an

improved endosomal escape motif is introduced into the light

chain variable region of a conventional therapeutic antibody

would be reduced or eliminated.

FIG. 28c shows a graph quantitatively comparing the

number of cells that taken up trypan blue at acidic pH by an

intact IgG-format RAS-targeting cytosol-penetrating antibody

in which an improved endosomal escape motif is introduced into the light-chain variable region of a conventional therapeutic antibody.

FIG. 29a shows the results of ELISA performed to measure

the affinities of an intact IgG-format RAS-targeting cytosol

penetrating antibody, in which an improved endosomal escape

motif is introduced into the light-chain variable region of a

conventional therapeutic antibody, for GppNHp-bound K-RAS

G12D and GDP-bound K-RAS G12D, which are K-RAS mutants.

FIG. 29b shows a schematic view showing a process of

constructing an intact IgG-format RAS-targeting cytosol

penetrating antibody in which an improved endosomal escape

motif is introduced into the RGD10 peptide-fused light-chain

variable region of a conventional therapeutic antibody.

FIG. 29c shows the results of confocal microscopy

performed to examine whether an intact IgG-format RAS

targeting cytosol-penetrating antibody in which an improved

endosomal escape motif is introduced into the RGD10 peptide

fused light-chain variable region of a conventional

therapeutic antibody would merge with intracellular activated

H-RAS G12V mutants.

FIG. 30a shows a process of constructing a cytosol

penetrating antibody having a light-chain variable region

from which endosomal escape ability has been removed and a

heavy-chain variable region into which an improved endosomal

escape motif has been introduced.

FIG. 30b shows a graph quantitatively comparing the

number of cells that have taken up trypan blue depending on

pH by a cytosol-penetrating antibody having a light-chain

variable region from which endosomal escape ability is

removed and a heavy-chain variable region into which an

improved endosomal escape motif is introduced.

FIG. 30c shows the results of confocal microscopy

performed to observe the GFP fluorescence by enhanced split

GFP complementation of a GFP11-SBP2-fused cytosol-penetrating

antibody having a light-chain variable region from which

endosomal escape ability has been removed, and a heavy-chain

variable region into which an improved endosomal escape motif

has been introduced.

FIG. 30d shows the results of confocal microscopy

performed using calcein in order to observe the cytosolic

localization of a cytosol-penetrating antibody having a

light-chain variable region from which endosomal escape

ability has been removed and a heavy-chain variable region

into which an improved endosomal escape motif has been

introduced.

FIG. 31a is a graph quantitatively comparing the number

of cells that taken up trypan blue depending on pH by mutants

constructed by substituting the 1 't amino acid glutamic acid

of the heavy-chain variable region (VH) of a cytosol penetrating antibody, which induces a change in properties of the antibody at acidic pH 5.5, with various amino acids.

FIG. 31b is a graph quantitatively comparing the number

of cells that taken up trypan blue depending on pH by mutants

constructed by substituting 1 0 2 "d amino acid leucine of the

heavy-chain variable region (VH) of a cytosol-penetrating

antibody, which induces a change in properties of the

antibody at acidic pH 5.5, with various amino acids.

FIG. 32a shows a graph quantitatively comparing the

number of cells that have taken up trypan blue depending on

pH by intact IgG-format cytosol-penetrating antibodies having

a light-chain variable region and/or a heavy-chain variable

region introduced with an endosomal escape motif having three

tryptophan residues.

FIG. 32b shows a bar graph showing the results of

observing the cytosolic localization of intact IgG-format

cytosol-penetrating antibodies having a light-chain variable

region and/or a heavy-chain variable region introduced with

an endosomal escape motif having three tryptophan residues by

confocal microscopy using calcein and quantifying the calcein

fluorescence of the confocal micrographs.

FIG. 33a shows a schematic view showing a process of

constructing an intact IgG-format cytosol-penetrating

antibody in which an improved endosomal escape motif has been

introduced into a heavy-chain variable region thereof and an improved endosomal escape motif has been introduced into a light-chain variable region of a conventional therapeutic antibody fused with an EpCAM-targeting peptide.

FIG. 33b shows a bar graph showing the results of

observing the cytosolic localization of an intact IgG-format

cytosol-penetrating antibody, in which an improved endosomal

escape motif has been introduced into a heavy-chain variable

region thereof and an improved endosomal escape motif has

been introduced into a light-chain variable region of a

conventional therapeutic antibody fused with an EpCAM

targeting peptide, by confocal microscopy using calcein and

quantifying the calcein fluorescence of the confocal

micrographs.

FIG. 33c shows a graph quantitatively comparing the

number of cells that have taken up trypan blue depending on

pH by an intact IgG-format cytosol-penetrating antibody in

which an improved endosomal escape motif has been introduced

into a heavy-chain variable region thereof and an improved

endosomal escape motif has been introduced into a light-chain

variable region of a conventional therapeutic antibody fused

with an EpCAM-targeting peptide.

FIG. 34a is a schematic view showing a process of

constructing an intact IgG-format cytosol-penetrating

antibody in which an improved endosomal escape motif has been introduced into the heavy-chain variable region of a conventional therapeutic antibody.

FIG. 34b is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by an

intact IgG-format cytosol-penetrating antibody in which an

improved endosomal escape motif has been introduced into the

heavy-chain variable region of a conventional therapeutic

antibody.

FIG. 35 is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by an

intact IgG-format cytosol-penetrating antibody comprising a

light-chain variable region and/or a heavy-chain variable

region introduced with aspartic acid.

FIG. 36a shows the results of observing a crystal of CT

59 Fab, formed under Index G1 conditions, by RI1000 (Rock

Imager1000; automatic protein crystal image analysis system).

FIG. 36b shows the three-dimensional structure of CT-59

refined and validated using the pymol program. The 1st amino

acid aspartic acid (D) and 9 5 th amino acid methionine (M) are

shown in yellow, and the 9 2 nd to 94th amino acids are shown in

orange.

BEST MODE FOR CARRYING OUT THE INVENTION

Unless defined otherwise, all the technical and

scientific terms used herein have the same meaning as those

generally understood by one of ordinary skill in the art to which the invention pertains. Generally, the nomenclature used herein and the experiment methods, which will be described below, are those well known and commonly employed in the art.

In one aspect, the present disclosure is directed to a

cytosol-penetrating antibody or an antigen-binding fragment

thereof comprising a light-chain variable region and/or

heavy-chain variable region that comprises a sequence

represented by the following formula in its CDR3:

X1-X2-X3-Z1

wherein X1-X2-X3 is an endosomal escape motif, and each

of Xl, X2 and X3 is selected from the group consisting of

tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine

(F);

Z1 is selected from the group consisting of methionine

(M), isoleucine (I), leucine (L), histidine (H), aspartic

acid (D), and glutamic acid (E);

the light-chain variable region and/or heavy-chain

variable region comprising Z1 induces a change in properties

of the antibody under endosomal acidic pH conditions; and

the antibody exhibits an ability to escape from

endosomes into the cytosol through the change in properties

of the antibody.

"Endosomal escape" in the present disclosure may mean

actively penetrating living cells by endocytosis, and then escaping from endosomes into the cytosol under acidic conditions.

"Endosomal escape motif" in the present disclosure

includes a one-dimensional structure comprising a specific

amino acid sequence having the property of inducing endosomal

escape under acidic conditions, and a three-dimensional

structure formed thereby. "Endosomal escape motif" may be

used interchangeably with "motif having endosomal escape

ability". An antibody comprising a light-chain variable

region (VL) or heavy-chain variable region (VH) that

comprises an "endosomal escape motif" is capable of

penetratingg the cytosol". "Cytosol-penetrating antibody"

means that an antibody that penetrated cells by endocytosis

escapes from endosomes into the cytosol under acidic

conditions. "Cytosol-penetrating antibody" may be used

interchangeably with "antibody having cytosol-penetrating

ability".

In the present disclosure, Z1 included in the endosomal

escape motif X1-X2-X3-Z1 may be located at the 9 5 th amino acid

of the light-chain variable region or the 102nd amino acid of

the heavy-chain variable region, as numbered by the Kabat

numbering system, and is the hydrophobic amino acid

methionine (M), isoleucine (I) or leucine (L), the negatively

charged amino acid aspartic acid (D) or glutamic acid (E), or

the positively charged amino acid histidine (H) . The 1st amino acid of the light-chain variable region or heavy-chain variable region of the cytosol-penetrating antibody according to the present disclosure can interact with negatively charged aspartic acid (D) or glutamic acid (E) and Z1, which is the 9 5 th amino acid of the light-chain variable region or the 1 0 2 nd amino acid of the heavy-chain variable region, under endosomal acidic pH conditions, thereby inducing a change in the properties of the antibody and allowing the antibody to have the ability to escape from endosomes into the cytosol.

In the present disclosure, the 1st amino acid of the

light-chain variable region and/or heavy-chain variable

region of the cytosol-penetrating antibody of the cytosol

penetrating antibody or antigen-binding fragment thereof may

interact with Z1 under endosomal acidic pH conditions to

induce a change in properties of the cytosol-penetrating

antibody.

In addition, as pH 7.4 changes to endosomal acidic pH

5.5, the interaction between Z1 of the endosomal escape motif

and the 1st amino acid of the light-chain variable region

and/or heavy-chain variable region changes. Namely, when Z1

is composed of the hydrophobic amino acid methionine (M),

isoleucine (I) or leucine (L) or the negatively charged amino

acid aspartic acid (D) or glutamic acid (E), the carboxylic

acid in the side chain of the negatively charged amino acid

becomes hydrophobic by partial protonation under the acidic conditions, and thus Z1 hydrophobically interacts with aspartic acid (D) or glutamic acid (E) , which is the 1st amino acid of the light-chain variable region or heavy-chain variable region.

In addition, regarding induction of a pH-dependent

change in properties of the antibody by interaction between

Z1 of the endosomal escape motif and the 1st amino acid of the

light-chain variable region or heavy-chain variable region,

when Z1 is composed of the hydrophobic amino acid methionine

(M), isoleucine (I) or leucine (L), it does not interact with

the negatively charged amino acid aspartic acid (D) or

glutamic acid (E), which is the 1st amino acid of the light

chain variable region or heavy-chain variable region, under

neutral pH conditions. However, as pH decreases, the

negatively charged amino acid becomes hydrophobic by

protonation, and thus hydrophobically interacts with Z1. As a

result, the distance between the two amino acids becomes

closer, thereby inducing a change in the structure and

function of the protein. This phenomenon is known as the

Tanford transition.

In addition, when Z1 is composed of histidine (H), as pH

changes from 7.4 to 5.5, the net charge of the amino acid

side chains becomes positive, and Z1 electrostatically

interacts with aspartic acid (D) or glutamic acid (E), which is the 1st amino acid of the light-chain variable region or heavy-chain variable region.

In an example of the present disclosure, in order to

confirm whether a pH-dependent change in the properties of

the antibody is induced by a pair of the 1st and 9 5 th amino

acids of the light-chain variable region, endosomal escape

ability was analyzed using alanine substitution mutants. As a

result, the alanine substitution mutations showed no pH

dependent endosomal escape ability. In addition, endosomal

escape ability was analyzed using mutations obtained by

substituting the 9 5 th amino acid with 20 different amino acids,

and as a result, mutants in which the 95th amino acid of the

light-chain variable region of the cytosol-penetrating

antibody according to the present disclosure is composed of

methionine (M), leucine (L), isoleucine (I), aspartic acid

(D), glutamic acid (E) and histidine (H) showed pH-dependent

endosomal escape ability.

In an example of the present disclosure, regarding a

pair of the 1st and 1 0 2 th amino acids of the heavy-chain

variable region, which induces a pH-dependent change in the

properties of the antibody, which has been found through the

alanine substitution mutation experiment in the same manner

as that in the above example, endosomal escape ability was

analyzed using mutations obtained by substituting the 102th

amino acid with 13 different amino acids, and as a result, mutants in which the 102th amino acid of the heavy-chain variable region of the cytosol-penetrating antibody according to the present disclosure is composed of methionine (M), leucine (L), isoleucine (I), aspartic acid (D), glutamic acid

(E) and histidine (H) showed pH-dependent endosomal escape

ability.

In addition, in one embodiment of the present disclosure,

the cytosol-penetrating antibody may further comprise,

between X3 and Z1, an amino acid sequence represented by (al

... -an) (where n is an integer ranging from 1 to 10). In one

embodiment of the present disclosure, when the cytosol

penetrating antibody further comprises, between X3 and Z1, an

amino acid sequence represented by (al-...-an) (where n is an

integer ranging from 1 to 10), a change in the properties of

the endosomal escape motif can be promoted while the length

of the CDR3 increases.

In the present disclosure, the endosomal escape motif

has a structure of X1-X2-X3-Z1 included in the light-chain

variable region; the heavy-chain variable region; or the

light-chain variable region and heavy-chain variable region,

and each of Xl, X2 and X3 is selected from the group

consisting of tryptophan (W), tyrosine (Y), histidine (H) and

phenylalanine (F).

In the present disclosure, the endosomal escape motif

X1-X2-X3 can react at intracellular endosomal weakly acidic pH, for example, a pH of 5.5 to 6.5, which is early endosomal pH, and thus Z1 can interact with the 1 st amino acid of the light-chain variable region or heavy-chain variable region, thereby changing the properties of the antibody and significantly increasing the endosomal escape efficiency of the antibody.

In the present disclosure, the endosomal escape motif Xl,

X2 and X3 are selected from the group consisting of amino

acids that easily interact with the hydrophilic head portion

and hydrophobic tail portion of 1-palmitoyl-2-oleoyl-sn

glycero-3-phosphatidylcholine (POPC) which is the major

phospholipid component of the inner endosomal membrane.

Specifically, the average binding activity of 20

different amino acids for 1-palmitoyl-2-oleoyl-sn-glycero-3

phosphatidylcholine (POPC) is higher in the order of

tryptophan (W), phenylalanine (F), tyrosine (Y), leucine (L),

isoleucine (I), cysteine (C), and methionine (M).

Specifically, the binding activity of 20 different amino

acids for the hydrophilic head portion of 1-palmitoyl-2

oleoyl-sn-glycero-3-phosphatidylcholine (POPC) is higher in

the order of arginine (R), tryptophan (W), tyrosine (Y),

histidine (H), asparagine (N), glutamine (Q), lysine (K),

and phenylalanine (F) . In addition, the binding activity of

20 different amino acids for the hydrophobic head portion of

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) is higher in the order of tryptophan (W), phenylalanine (F), leucine (L), methionine (M), isoleucine (I), valine (V), and tyrosine (Y).

In the present disclosure, amino acids constituting Xl,

X2 and X3 of the endosomal escape motif may include tyrosine

(Y) and histidine (H), which constitute a wild-type cytosol

penetrating antibody. Thus, these amino acids may include

tryptophan (W) and phenylalanine (F), which have a higher

average binding affinity than tyrosine (Y) for 1-palmitoyl-2

oleoyl-sn-glycero-3-phosphatidylcholine (POPC).

In an example of the present disclosure, amino acids

that easily interact with 1-palmitoyl-2-oleoyl-sn-glycero-3

phosphatidylcholine (POPC) was examined through literature

search. Furthermore, tryptophan (W) having high binding

affinity for the hydrophilic head portion and hydrophobic

tail portion was introduced into Xl, X2 and X3 of the

endosomal escape motif, and endosomal escape ability was

analyzed. As a result, an improved cytosol-penetrating

antibody according to the present disclosure showed a higher

pH-dependent endosomal escape ability than the wild-type

cytosol-penetrating antibody including tyrosine (Y), tyrosine

(Y) and histidine (H) in Xl, X2 and X3, respectively.

In another example, in order to examine whether

interaction with the head portion or tail portion of 1

palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) is important for endosomal escape, mutants were constructed by introducing arginine (R) which easily binds only to the head portion, isoleucine (I) which easily binds only to the tail portion, and glycine (G) which shows significantly low interaction with the lipid, into Xl, X2 and X3 of the endosomal escape motif, and endosomal escape ability was analyzed. As a result, two mutants, excluding a cytosol penetrating antibody introduced with tryptophan (W) according to the present disclosure, all showed significantly reduced endosomal escape ability. This suggests that interactions with the hydrophilic head and hydrophobic tail of the lipid are all involved in endosomal escape.

In still another example, the endosomal escape motif of

the light-chain variable region may comprise one or more

tryptophans, or one or two tryptophans.

In order to increase the effect of a substance that

exhibits its activity in the cytosol, the amount of the

substance located in the cytosol should ultimately increase.

Hence, studies have been conducted to increase endosomal

escape ability. Such studies have been conducted mainly on

cell-penetrating peptides (CPPs). In particular, interaction

with the lipid membrane is essential for passage through the

cell lipid membrane, a strategy for enhancing this

interaction has been introduced. As one example, tryptophan was added to the N-terminus of a cytosol-penetrating peptide rich in arginine and to the middle portion of the peptide.

However, this approach has not been attempted on

antibodies. Tryptophan (W) is an amino acid showing high

interaction with the hydrophilic head portion and hydrophobic

tail portion of 1-palmitoyl-2-oleoyl-sn-glycero-3

phosphatidylcholine (POPC) which is the major phospholipid

component of the cell membrane. Thus, it can improve

interaction with the inner endosomal membrane and induce

endosomal escape.

Specifically, in an example of the present disclosure,

the endosomal escape motif X1-X2-X3 of the light-chain

variable region and/or heavy-chain variable region may

comprise a sequence selected from the group consisting of W

W-W, W-W-H, W-Y-W, Y-W-W, W-Y-H, and Y-W-H (where W is

tryptophan, Y is tyrosine, H is histidine).

In an example of the present disclosure, it was found

that the endosomal escape motif X1-X2-X3 of the light-chain

variable region and/or heavy-chain variable region increases

the endosomal escape ability through a change in the

properties of the antibody by induction of the interaction

under endosomal acidic pH conditions.

As used herein, the term "endosomal acidic pH" refers to

a pH range of 6.0 to 4.5, which satisfies early endosomal and

late endosomal pH conditions and in which the side-chain properties of aspartic acid (D) and glutamic acid (E) may change.

The CDR3 of the light-chain variable region comprising

the endosomal escape motif may comprise one or more sequences

selected from the following group consisting of:

QQYWWHMYT (SEQ ID NO: 8);

QQYWYWMYT (SEQ ID NO: 9);

QQYYWWMYT (SEQ ID NO: 10);

QQYWYHMYT (SEQ ID NO: 11);

QQYYWHMYT (SEQ ID NO: 12); and

QQYWWWMYT (SEQ ID NO: 51).

The light-chain variable region comprising the endosomal

escape motif may comprise a sequence having a homolog of at

least 80%, for example, 85%, 90%, 95%, 96%, 97%, 98%, 99% or

100%, to a light-chain variable region sequence selected from

the group consisting of, for example, SEQ ID NOS: 1 to 5, 13

to 23, 25 to 37, 50, and 60 to 64.

Improved endosomal escape efficiency can also be

achieved at the same level even when the endosomal escape

motif is included in the heavy-chain variable region.

Specifically, the heavy-chain variable region may include Xl

X2-X3-Z1 (wherein each of Xl, X2 and X3 is selected from the

group consisting of tryptophan (W), tyrosine (Y), histidine

(H) and phenylalanine (F)) in its CDR3, and Z1 can interact

with the 1st amino acid of the heavy-chain variable region under endosomal acidic pH conditions, thus changing the properties of the cytosol-penetrating antibody and enabling the antibody to have the ability to escape from endosomes into the cytosol.

The CDR3 of the heavy-chain variable region comprising

the endosomal escape motif may comprise one or more sequences

selected from the following group consisting of SEQ ID NOS: 46

to 49, and 53:

GWYWMDL (SEQ ID NO: 46);

GWYWFDL (SEQ ID NO: 47);

GWYWGFDL (SEQ ID NO: 48);

YWYWMDL (SEQ ID NO: 49); and

GWWWMDL (SEQ ID NO: 53).

the light-chain variable region comprise a sequence

having a homolog of at least 80% to a light-chain variable

region sequence selected from the group consisting of SEQ ID

NOS: 1 to 5, 13 to 23, 25 to 37, 50, and 60 to 64.

The heavy-chain variable region comprising the endosomal

escape motif may comprise a sequence having a homolog of at

least 80%, for example, 85%, 90%, 95%, 96%, 97%, 98%, 99% or

100%, to a heavy-chain variable region sequence selected from

the group consisting of, for example, SEQ ID NOS: 39 to 42,

52, and 54 to 59.

In addition, in one embodiment, the sequence may further

comprise Z2 linked to Xl, and thus may be represented by the

following formula:

Z2-X1-X2-X3-Z1,

wherein Z2 is selected from the group consisting of

glutamine (Q), leucine (L) histidine (H).

As described above, the sequence is represented by Z2

X1-X2-X3-Z1, the 1st amino acid of the light-chain variable

region and/or heavy-chain variable region interacts with Z1

and/or Z2 to induce pH-dependent endosomal escape under

endosomal acidic pH conditions.

The CDR3 of the light-chain variable region comprising

the endosomal escape motif may comprise a sequences of SEQ ID

NO: 24 as set forth below:

QHYWYWMYT (SEQ ID NO: 24).

As used herein, "antibody" is meant to include an intact

antibody form that specifically binds to a target as well as

an antigen-binding fragment of the antibody.

The complete antibody is a structure having two full

length light chains and two full-length heavy chains, and

each light chain is linked by a disulfide bond with a heavy

chain. A constant region of the heavy chain has gamma (y), mu

(p), alpha (a), delta (5), and epsilon (E) types. Sub-classes

have gamma 1 (yl) , gamma 2 (y 2 ) , gamma 3 (y 3 ) , gamma 4 (y4) ,

alpha

1 (al), and alpha 2 (a2) types. A constant region of the light

chain has kappa (K) and lambda (X) types.

An antigen binding fragment or an antibody fragment of

an antibody refers to a fragment having an antigen binding

function and includes Fab, F(ab'), F(ab')2, Fv, and the like.

Fab of the antibody fragments has a structure including

variable regions of a light chain and a heavy chain, a

constant region of the light chain, and a first constant

region (CH1 domain) of the heavy chain with one antigen

binding site. Fab' differs from Fab in that it has a hinge

region containing one or more cysteine residues at the C

terminal of the heavy chain CH1 domain. The F(ab')2 antibody

is produced when the cysteine residue of the hinge region of

the Fab' forms a disulfide bond. Recombinant techniques for

generating Fv fragments with minimal antibody fragments

having only a heavy-chain variable region and a light-chain

variable region are described in PCT International

Publication Nos. W088/001649, W088/006630, W088/07085,

W088/07086, and W088/09344. A two-chain Fv has a non-covalent

bonding between a heavy-chain variable region and a light

chain variable region. A single chain Fv (scFv) is connected

to a heavy-chain variable region and a light-chain variable

region via a peptide linker by a covalent bond or directly at

the C-terminal. Thus, the single chain Fv (scFv) has a

structure such as a dimer like the two-chain Fv. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, when the whole antibody is cleaved with papain, Fab can be obtained, and when whole antibody is cut with pepsin, F(ab')2 fragment can be obtained), and it can also be produced through gene recombinant technology.

In one embodiment, the antibody according to the present

disclosure may be an Fv form (e.g., scFv) or a whole antibody

form. The cytosol-penetrating antibody according to the

present disclosure may be an IgG, IgM, IgA, IgD or IgE type.

For example, it may be an IgG1, IgG2, IgG3, IgG4, IgM, IgE,

IgAl, IgA5, or IgD type. Most preferably, it may be an

intact IgG-format monoclonal antibody.

Further, the heavy chain constant region can be selected

from any one isotype of gamma (y), mu (p), alpha (a), delta

(5), and epsilon (s). Sub-classes have gamma 1 (yl), gamma 2

(y2), gamma 3 (y3), gamma 4 (y4), alpha 1 (al), and alpha 2

(a2) types. A constant region of the light chain has kappa

(K) and lambda (X) types.

The term "heavy chain" as used herein refers to a full

length heavy chain and fragments thereof including a variable

region domain VH including an amino acid sequence with

sufficient variable region sequence to confer specificity to

an antigen and three constant region domains CH1, CH2, and

CH3. The term "light chain" as used herein refers to a full

length heavy chain and fragments thereof including a variable region domain VL including an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen and a constant region domain CL.

In the present disclosure, the antibody includes

monoclonal antibodies, multispecific antibodies, human

antibodies, humanized antibodies, chimeric antibodies,

single-chain Fvs (scFV), single chain antibodies, Fab

fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and

anti-idiotype (anti-Id) antibodies, and epitope-binding

fragments of these antibodies, but is not limited thereto.

An "Fv" fragment is an antibody fragment that contains

complete antigen recognition and binding sites. Such region

includes a heavy chain variable domain and a light chain

variable domain, for example, dimers substantially tightly

covalently associated with scFv.

"Fab" fragment contains the variable and constant domain

of the light-chain and the variable and first constant domain

(CH1) of the heavy chain. F(ab')2 antibody fragment generally

includes a pair of Fab fragments covalently linked by hinge

cysteine near their carboxy-terminus.

"Single chain Fv" or "scFv" antibody fragment comprises

VH and VL domains of the antibody. Such domains are within a

single polypeptide chain. The Fv polypeptide may further

include a polypeptide linker between the VH domain and the VL domain such that the scFv can form the desired structure for antigen binding.

The monoclonal antibody refers to an antibody obtained

from a substantially homogeneous population of antibodies,

i.e., the same except for possible naturally occurring

mutations that may be present in trace amounts of individual

antibodies that occupy the population. The monoclonal

antibody is highly specific and is derived against a single

antigenic site.

The non-human (e.g. murine) antibody of the "humanized"

form is a chimeric antibody containing minimal sequence

derived from non-human immunoglobulin. In most cases, the

humanized antibody is a human immunoglobulin (receptor

antibody) that has been replaced by a residue from the

hypervariable region of a non-human species (donor antibody),

such as a mouse, rat, rabbit, and non-human primate, having

specificity, affinity, and ability to retain a residue from

the hypervariable region of the receptor.

"Human antibody" is a molecule derived from human

immunoglobulin and means that all of the amino acid sequences

constituting the antibody including the complementarity

determining region and the structural region are composed of

human immunoglobulin.

A heavy chain and/or light chain is partly identical or

homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) are identical or homologous to corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass "chimeric" antibodies (immunoglobulins) as well as a fragment of such antibody exhibiting the desired biological activity.

"Antibody variable domain" as used herein refers to the

light and heavy chain regions of an antibody molecule

including the amino acid sequences of a complementarity

determining region (CDR; i.e., CDR1, CDR2, and CDR3) and a

framework region (FR). VH refers to a variable domain of the

heavy chain. VL refers to a variable domain of the light

chain.

"Complementarity determining region" (CDR; i.e., CDR1,

CDR2, and CDR3) refers to the amino acid residue of the

antibody variable domain, which is necessary for antigen

binding. Each variable domain typically has three CDR regions

identified as CDR1, CDR2, and CDR3.

"Framework region" (FR) is a variable domain residue

other than a CDR residue. Each variable domain typically has

four FRs identified as FR1, FR2, FR3, and FR4.

In another aspect, the present disclosure is directed to

a composition for delivering an active substance into cytosol, comprising the cytosol-penetrating antibody or antigen binding fragment thereof.

The active substance may be a type fused or bonded to

the antibody, and the active substance may be one or more

selected from the group consisting of, for example, peptides,

proteins, toxins, antibodies, antibody fragments, RNAs,

siRNAs, DNAs, small molecule drugs, nanoparticles, and

liposomes, but is not limited thereto.

The proteins may be antibodies, antibody fragments,

immuoglubulin, peptides, enzymes, growth factors, cytokines,

transcription factors, toxins, antigen peptides, hormones,

carrier proteins, motor function proteins, receptors,

signaling proteins, storage proteins, membrane proteins,

transmembrane proteins, internal proteins, external proteins,

secretory proteins, viral proteins, glycoproteins, cleaved

proteins, protein complexes, chemically modified proteins, or

the like.

The RNA or ribonucleic acid is based on ribose, a kind

of pentose, is a kind of nucleic acid consisting of a chain

of nucleotides, has a single-stranded structure, and is

formed by transcription of a portion of DNA. In one

embodiment, the RNA may be selected from the group consisting

of rRNA, mRNA, tRNA, miRNA, snRNA, snoRNA, and aRNA, but is

not limited thereto.

The siRNA (Small interfering RNA) is a small RNA

interference molecule composed of dsRNA, and functions to

bind to and degrade an mRNA having a target sequence. It is

used as a disease treating agent or has an activity of

inhibiting expression of a protein translated from a target

mRNA by degrading the target mRNA. Due to this activity, it

is widely used herein.

The DNA or deoxyribonucleic acid is a kind of nucleic

acid, is composed of a backbone chain comprising

monosaccharide deoxyribose linked by phosphate, together with

two types of nucleobases (purines and pyrimidines), and

stores the genetic information of cells.

As used herein, the term "small-molecule drugs" refers

to organic compounds, inorganic compounds or organometallic

compounds that have a molecular weight of less than about

1000 Da and are active as therapeutic agents against diseases.

The term is used in a broad sense herein. The small-molecule

drugs herein encompass oligopeptides and other biomolecules

having a molecular weight of less than about 1000 Da.

In the present disclosure, a nanoparticle refers to a

particle including substances ranging between 1 and 1,000 nm

in diameter. The nanoparticle may be a metal nanoparticle, a

metal/metal core shell complex consisting of a metal

nanoparticle core and a metal shell enclosing the core, a

metal/non-metal core shell consisting of a metal nanoparticle core and a non-metal shell enclosing the core, or a non metal/metal core shell complex consisting of a non-metal nanoparticle core and a metal shell enclosing the core.

According to an embodiment, the metal may be selected from

gold, silver, copper, aluminum, nickel, palladium, platinum,

magnetic iron and oxides thereof, but is not limited thereto,

and the non-metal may be selected from silica, polystyrene,

latex and acrylate type substances, but is not limited

thereto.

In the present disclosure, liposomes include at least

one lipid bilayer enclosing the inner aqueous compartment,

which is capable of being associated by itself. Liposomes may

be characterized by membrane type and size thereof. Small

unilamellar vesicles (SUVs) may have a single membrane and

may range between 20 and 50 nm in diameter. Large unilamellar

vesicles (LUVs) may be at least 50 nm in diameter.

Oliglamellar large vesicles and multilamellar large vesicles

may have multiple, usually concentric, membrane layers and

may be at least 100 nm in diameter. Liposomes with several

nonconcentric membranes, i.e., several small vesicles

contained within a larger vesicle, are referred to as

multivesicular vesicles.

The term "fusion" or "binding" refers to unifying two

molecules having the same or different function or structure,

and the methods of fusing may include any physical, chemical or biological method capable of binding the tumor tissue penetrating peptide to the protein, small-molecule drug, nanoparticle or liposome. Preferably, the fusion may be made by a linker peptide, and for example, the linker peptide may mediate the fusion with the bioactive molecules at various locations of an antibody light-chain variable region of the present disclosure, an antibody, or fragments thereof.

In still another aspect, the present disclosure provides

a pharmaceutical composition for prevention or treatment of

cancer, comprising: the above-described cytosol-penetrating

antibody or antigen binding fragment thereof; and an active

substance to be delivered into cytosol by the cytosol

penetrating antibody or antigen binding fragment thereof.

The use of the active substance can impart the property

of penetrating cells and localizing in the cytosol, without

affecting the high specificity and affinity of antibodies for

antigens, and thus can localize in the cytosol which is

currently classified as a target in disease treatment based

on small-molecule drugs, and at the same time, can exhibit

high effects on the treatment and diagnosis of tumor and

disease-related factors that show structurally complex

interactions through a wide and flat surface between protein

and protein.

The use of the pharmaceutical composition for prevention

or treatment of cancer can impart the property of enabling the antibody to penetrate cells and remain in the cytosol, without affecting the high specificity and affinity of the antibody for antigens, and thus the antibody can localize in the cytosol which is currently classified as a target in disease treatment based on small-molecule drugs, and at the same time, can be expected to exhibit high effects on the treatment and diagnosis of tumor and disease-related factors that show structurally complex interactions through a wide and flat surface between protein and protein.

In one example of the present disclosure, the

pharmaceutical composition can selectively inhibit KRas

mutants, which are major drug resistance-associated factors

in the use of various conventional tumor therapeutic agents,

and at the same time, can be used in combination with

conventional therapeutic agents to thereby exhibit effective

anticancer activity.

The cancer may be selected from the group consisting of

squamous cell carcinoma, small cell lung cancer, non-small

cell lung cancer, adenocarcinoma of lung, squamous cell

carcinoma of lung, peritoneal cancer, skin cancer, skin or

ocular melanoma, rectal cancer, anal cancer, esophageal

cancer, small intestine cancer, endocrine cancer, parathyroid

cancer, adrenal cancer, soft tissue sarcoma, urethral cancer,

chronic or acute leukemia, lymphoma, hepatoma,

gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, liver cancer and head and neck cancer.

When the composition is prepared as a pharmaceutical

composition for preventing or treating cancer or

angiogenesis-related diseases, the composition may include a

pharmaceutically acceptable carrier. The pharmaceutically

acceptable carrier contained in the composition is typically

used in the formulation. Examples of the pharmaceutically

acceptable carrier included in the composition may include,

but are not limited to, lactose, dextrose, sucrose, sorbitol,

mannitol, starch, acacia rubber, calcium phosphate, alginate,

gelatin, calcium silicate, minute crystalline cellulose,

polyvinyl pyrrolidone, cellulose, water, syrup, methyl

cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate,

talc, magnesium stearate and mineral oil, etc., but are not

limited thereto. In addition to the above ingredients, the

pharmaceutical composition may further include a lubricant, a

wetting agent, a sweetener, a flavoring agent, an emulsifier,

a suspension, a preservative, etc.

The pharmaceutical composition for preventing or

treating cancer or angiogenesis-related diseases may be

administered orally or parenterally. Such a parenteral administration includes intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, nasal administration, intrapulmonary administration, intrarectal administration, etc. Because a protein or peptide is digested when administered orally, it is preferred that a composition for oral administration is formulated to coat an active substance or to be protected against degradation in stomach.

Also, the pharmaceutical composition may be administered by

any device which can transport active substances to target

cells.

Proper dose of the pharmaceutical composition for

preventing or treating cancer or angiogenesis-related

diseases may vary according to various factors such as method

for formulating, administration method, age, weight, gender,

pathological state of patient, food, administration time,

administration route, excretion rate and reaction sensitivity,

etc. Preferably, a proper dose of the composition is within

the range of 0.001 and 100 mg/kg based on an adult. The term

"pharmaceutically effective dose" as used herein refers to an

amount sufficient to prevent or treat cancer or angiogenesis

related diseases.

The composition may be formulated with pharmaceutically

acceptable carriers and/or excipients according to a method

that can be easily carried out by those skilled in the art, and may be provided in a unit-dose form or enclosed in a multiple-dose vial. Here, the formulation of the pharmaceutical composition may be in the form of a solution, a suspension, syrup or an emulsion in oily or aqueous medium, or may be extracts, powders, granules, tablets or capsules, and may further include a dispersion agent or a stabilizer.

Also, the composition may be administered individually or in

combination with other therapeutic agents, and may be

administered sequentially or simultaneously with conventional

therapeutic agents. Meanwhile, the composition includes an

antibody or an antigen-binding fragment, and thus may be

formulated into immuno liposome. Liposome including an

antibody may be prepared according to a method well known in

the pertinent art. The immuno liposome is a lipid composition

including phosphatidylcholine, cholesterol and

polyethyleneglycol-derived phosphatidylethanolamine, and may

be prepared by reverse phase evaporation method. For example,

a Fab' fragment of antibody may be conjugated to liposome

through disulphide exchange reaction. Liposome may further

include chemical therapeutic agents such as Doxorubicin.

In yet another aspect, the present disclosure is

directed to a pharmaceutical composition for diagnosis of

cancer, comprising: the above-described cytosol-penetrating

antibody or antigen binding fragment thereof; and an active substance to be delivered into cytosol by the cytosol penetrating antibody or antigen binding fragment thereof.

The term "diagnosis" as used herein refers to

demonstrating the presence or characteristic of a

pathophysiological condition. Diagnosing in the present

disclosure refers to demonstrating the onset and progress of

cancer.

The intact immunoglobulin-format antibody and a fragment

thereof may bind to a fluorescent substance for molecular

imaging in order to diagnose cancer through images.

The fluorescent substance for molecular imaging refers

to all substances generating fluorescence. Preferably, red

or near-infrared fluorescence is emitted, and more preferably,

a fluorescence with high quantum yield is emitted. However,

the fluorescence is not limited thereto.

Preferably, the fluorescent substance for molecular

imaging is a fluorescent substance, a fluorescent protein or

other substances for imaging, which may bind to the tumor

tissue-penetrating peptide that specifically binds to the

intact immunoglobulin-format antibody and a fragment thereof,

but is not limited thereto.

Preferably, the fluorescent substance is fluorescein,

BODYPY, tetramethylrhodamine, Alexa, cyanine, allopicocyanine,

or a derivative thereof, but is not limited thereto.

Preferably, the fluorescent protein is Dronpa protein,

enhanced green fluorescence protein (EGFP), red fluorescent

protein (DsRFP), Cy5.5, which is a cyanine fluorescent

substance presenting near-infrared fluorescence, or other

fluorescent proteins, but is not limited thereto.

Preferably, other substances for imaging are ferric

oxide, radioactive isotope, etc., but are not limited thereto,

and they may be applied to imaging equipment such as MR, PET.

In a further another aspect, the present disclosure is

directed to a nucleic acid encoding the above-described

antibody or antigen-binding fragment thereof.

The nucleic acid is a polynucleotide, and the term

"polynucleotide" as used herein refers to a

deoxyribonucleotide or ribonucleotide polymer present in a

single-stranded or double-stranded form. It includes RNA

genome sequence, DNA (gDNA and cDNA), and RNA sequence

transcribed therefrom. Unless otherwise described, it also

includes an analog of the natural polynucleotide.

The polynucleotide includes not only a nucleotide

sequence encoding the above-described light-chain variable

region (VL) and heavy-chain variable region (VH) having

improved endosomal escape ability, but also a complementary

sequence thereto. The complementary sequence includes a

sequence fully complementary to the nucleotide sequence and a

sequence substantially complementary to the nucleotide sequence. For example, this complementary sequence may include a sequence that may be hybridized with a nucleotide sequence encoding a light-chain variable region (VL) and heavy-chain variable region (VH) having any one sequence selected from the group consisting of SEQ ID NOS: 1 to 5, 13 to 23, 25 to 37, 50, and 60 to 64, and SEQ ID NOS: 39 to 42,

52, and 54 to 59 under stringent conditions known in the

pertinent art.

The polynucleotide includes not only a nucleotide

sequence encoding the above-described light-chain region

(kds), but also a complementary sequence thereto. The

complementary sequence includes a sequence fully

complementary to the nucleotide sequence and a sequence

substantially complementary to the nucleotide sequence. For

example, this means a sequence that may be hybridized with a

nucleotide sequence encoding an amino acid sequence of any

one of SEQ ID NO:1 to SEQ ID NO: 3 under stringent conditions

known in the pertinent art.

The nucleic acid may be modified. The modification

includes the addition, deletion, or non-conservative

substitution or conservative substitution of nucleotides. The

nucleic acid encoding the amino acid sequence is interpreted

to include a nucleotide sequence that has a substantial

identity to the nucleotide sequence. The substantial identity

may refer to a sequence having a homology of at least 80%, a homology of at least 90%, or a homology of at least 95% when aligning the nucleotide sequence to correspond to any other sequence as much as possible and analyzing the aligned sequence using an algorithm generally used in the pertinent art.

The DNA encoding the antibody can be easily separated or

synthesized using conventional procedures (for example, using

an oligonucleotide probe capable of specifically binding to

DNA encoding the heavy chain and the light chain of the

antibody).

In a still further aspect, the present disclosure is

directed to a method for producing the above-described

cytosol-penetrating antibody or antigen binding fragment

thereof, comprising a step of grafting the endosomal escape

motif X1-X2-X3-Z1 (wherein X1-X2-X3 is selected from the

group consisting of tryptophan (W), tyrosine (Y), histidine

(H), and phenylalanine (F)) into the CDR3 of a light chain

and/or heavy-chain variable region.

The present disclosure can provide an antibody or

antigen-binding fragment thereof having a cytosol-penetrating

ability by substituting the light-chain variable region (VL)

of a conventional antibody with a light-chain variable region

(VL) having improved endosomal escape ability and

substituting the heavy-chain variable region (VH) of the conventional antibody with a heavy-chain variable region (VH) having improved endosomal escape ability.

In one embodiment, a method of producing an intact

immunoglobulin-format antibody, which penetrates cells and

localizes in the cytosol, by use of a cytosol-penetrating

light-chain variable region (VL) having improved endosomal

escape ability and a cytosol-penetrating heavy-chain variable

region having endosomal escape ability, comprises the steps

of: obtaining a nucleic acid, in which a light-chain variable

region (VL) in a light chain comprising the light-chain

variable region (VL) and a light chain constant region is

substituted with a light-chain variable region (VL) having

endosomal escape ability or a heavy-chain variable region

(VH) and a heavy chain constant region (CH) are substituted

with a heavy-chain variable region (VH) having endosomal

escape ability, cloning the nucleic acid into a vector, and

transforming the vector into a host cell to express the

antibody or an antigen binding fragment thereof; and

recovering the expressed antibody or an antigen binding

fragment thereof.

The above-described method makes it possible to produce

an intact immunoglobulin-format antibody having increased

endosomal escape ability and cytosol-penetrating ability.

Furthermore, transformation with a vector expressing a heavy

chain comprising a heavy-chain variable region capable of recognizing a specific protein in cells makes it possible to express an antibody which is able to penetrate cells and localize in the cytosol to bind to the specific protein. The vector may be either a vector system that co-expresses the heavy chain and the light chain in a single vector or a vector system that expresses the heavy chain and the light chain in separate vectors. In the latter case, the two vectors may be introduced into a host cell by co transformation and targeted transformation.

In the present disclosure, the vector may be either a

vector system that co-expresses the heavy chain and the light

chain in a single vector or a vector system that expresses

the heavy chain and the light chain in separate vectors. In

the latter case, the two vectors may be introduced into a

host cell by co-transformation and targeted transformation.

The term "vector" as used herein refers to a means for

expressing a target gene in a host cell. For example, the

vector may include plasmid vector, cosmid vector,

bacteriophage vector, and virus vectors such as adenovirus

vector, retrovirus vector, and adeno-associated virus vector.

The vector that may be used as the recombinant vector may be

produced by operating plasmid (for example, pSC101, pGV1106,

pACYC177, ColEl, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9,

pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and

pUC19, etc.), phages (for example, Xgt4XB, X-Charon, XAzl and

M13, etc.), or virus (for example, CMV, SV40, etc.) commonly

used in the pertinent art.

The light-chain variable region, the light-chain

constant region (CL), the heavy-chain variable region (VH),

and the heavy-chain constant region (CH1-hinge-CH2-CH3) of

the present disclosure in the recombinant vector may be

operatively linked to a promoter. The term "operatively

linked" as used herein means a functional linkage between a

nucleotide expression control sequence (such as a promoter

sequence) and a second nucleotide sequence. Accordingly, the

control sequence may control the transcription and/or

translation of the second nucleotide sequence.

The recombinant vector may be generally constructed as a

vector for cloning or a vector for expression. As the vector

for expression, vectors generally used for expressing foreign

protein from plants, animals or microorganisms in the

pertinent art may be used. The recombinant vector may be

constructed by various methods known in the pertinent art.

The recombinant vector may be constructed to be a vector

that employs a prokaryotic cell or an eukaryotic cell as a

host. For example, when the vector used is an expression

vector and employs a prokaryotic cell as a host, the vector

generally includes a strong promoter which may promote

transcription (for example, pLX promoter, trp promoter, lac

promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and termination sequences for transcription/translation. When the vector employs an eukaryotic cell as a host, a replication origin operating in the eukaryotic cell included in the vector may include an fl replication origin, an SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an

AAV replication origin, a CMV replication origin and a BBV

replication origin, etc., but is not limited thereto. In

addition, a promoter derived from a genome of a mammal cell

(for example, a metalthionine promoter) or a promoter derived

from a virus of a mammal cell (for example, an adenovirus

anaphase promoter, a vaccinia virus 7.5K promoter, a SV40

promoter, a cytomegalo virus (CMV) promoter, or a tk promoter

of HSV) may be used, and the promoter generally has a

polyadenylated sequence as a transcription termination

sequence.

Another aspect of the present disclosure provides a host

cell transformed with the recombinant vector.

Any kind of host cell known in the pertinent art may be

used as a host cell. Examples of a prokaryotic cell include

strains belonging to the genus Bascillus such as E. coli

JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E.

coli X 1776, E. coli W3110, Bascillus subtilus and Bascillus

thuringiensis, Salmonella typhimurium, intestinal flora and

strains such as Serratia marcescens and various Pseudomonas

Spp., etc. In addition, when the vector is transformed in an

eukaryotic cell, a host cell such as yeast (Saccharomyce

cerevisiae), an insect cell, a plant cell, and an animal cell,

for example, SP2/0, CHO (Chinese hamster ovary) K1, CHO DG44,

PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RN, and MDCK

cell line, etc., may be used.

\Another aspect of the present disclosure may provide a

method for producing an intact immunoglobulin-format antibody

that penetrates cells and localizes in the cytosol, the

method comprising a step of culturing the above-described

host cell.

A recombinant vector may be inserted into a host cell

using an insertion method well known in the pertinent art.

For example, when a host cell is a prokaryotic cell, the

transfer may be carried out according to CaCl 2 method or an

electroporation method, etc., and when a host cell is an

eukaryotic cell, the vector may be transferred into a host

cell according to a microscope injection method, calcium

phosphate precipitation method, an electroporation method, a

liposome-mediated transformation method, and a gene

bombardment method, etc., but the transferring method is not

limited thereto. When using microorganisms such as E. coli,

etc. the productivity is higher than using animal cells.

However, although it is not suitable for production of intact

Ig form of antibodies due to glycosylation, it may be used for production of antigen binding fragments such as Fab and

Fv.

The method for selecting the transformed host cell may

be readily carried out according to a method well known in

the pertinent art using a phenotype expressed by a selected

label. For example, when the selected label is a specific

antibiotic resistance gene, the transformant may be readily

selected by culturing the transformant in a medium containing

the antibiotic.

EXAMPLES

Hereinafter, the present disclosure will be described in

further detail with reference to examples. It will be obvious

to a person having ordinary skill in the art that these

examples are illustrative purposes only and are not to be

construed to limit or change the scope of the present

disclosure.

Example 1: Expression and Purification of Cytosol

Penetrating Antibody (Cytotransmab)

In order to elucidate the endosomal escape mechanism of

a cytosol-penetrating antibody and to improve the endosomal

escape mechanism, the cytosol-penetrating antibody was

purified.

Specifically, in order to construct a heavy-chain

expression vector for producing an intact IgG-format monoclonal antibody, a DNA encoding a heavy chain comprising an antibody heavy-chain variable region (humanized hTO VH;

SEQ ID NO: 38) and a heavy-chain constant region (CH1-hinge

CH2-CH3), which has a secretion signal peptide-encoding DNA

fused to the 5' end, was cloned into a pcDNA3.4 vector

(Invitrogen) by NotI/HindIII.

Furthermore, in order to construct a vector that

expresses a light chain, a DNA encoding a light chain

comprising a cytosol-penetrating light-chain variable region

(hT4 VL; SEQ ID NO: 65) and light-chain constant region (CL),

which has a secretion signal peptide-encoding DNA fused to

the 5' end, was cloned into a pcDNA3.4 vector (Invitrogen) by

use of NotI/HindIII.

The light-chain and heavy-chain expression vectors were

transiently transfected, and the proteins were expressed and

purified. In a shaking flask, HEK293-F cells suspension

growing in serum-free FreeStyle 293 expression medium

(Invitrogen) were transfected with a mixture of plasmid and

polyethylenimine (PEI) (Polyscience). After 200 mL

transfection in a shaking flask (Corning), HEK293-F cells

were seeded into 100 ml of medium at a density of 2.0 x

106 cells/ml, and cultured at 150 rpm and in 8% C0 2 . To

produce each monoclonal antibody, a suitable heavy-chain and

light-chain plasmid were diluted in 10 ml of FreeStyle 293

expression medium (Invitrogen) (125 pg heavy chain, 125 pg light chain, a total of 250 pg (2.5 pg/ml)), and the dilution was mixed with 10 ml of medium containing 750 pg (7.5 pg/ml) of PEI, and the mixture was incubated at room temperature for

10 minutes. The incubated medium mixture was added to 100 ml

of the seeded cell culture which was then cultured at 150 rpm

in 8% C02 for 4 hours, after which 100 ml of FreeStyle 293

expression was added to the cell culture, followed by culture

for 6 days.

In accordance with the standard protocol, the protein

was purified from the collected cell culture supernatant. The

antibody was applied to a Protein A Sepharose column (GE

Healthcare), and washed with PBS (pH 7.4). The antibody was

eluted using 0.1 M glycine buffer (pH 3.0), and then

immediately neutralized with 1M Tris buffer. The eluted

antibody fraction was concentrated while the buffer was

replaced with PBS (pH 7.4) by dialysis. The purified protein

was quantified by measuring the absorbance at 280 nm and the

absorption coefficient.

Example 2: Observation of Trafficking after Endocytosis

of Cytosol-Penetrating Antibody

Trafficking from endocytosis of the developed cytosol

penetrating antibody to localization into the cytosol was

observed. This may be an important clue to the mechanism of

endosomal escape into the cytosol.

FIG. 1 shows of a pulse-chase experiment and confocal

microscopy observation performed to observe the transport

process and stability of the cytosol-penetrating antibody

(cytotransmab) TMab4 or cell-penetrating peptide TAT

introduced into cells.

Specifically, a cover slip was added to 24-well plates,

and 2.5 x 104 HeLa cells per well were added to 0.5 ml of 10%

FBS-containing medium and cultured for 12 hours under the

conditions of 5% CO2 and 37°C. When the cells were stabilized,

the cells were transiently transfected with pcDNA3.4-flag

rabl. To maximize the efficiency of transient transfection,

Opti-MEM media (Gibco) was used. 500 ng of pcDNA3.4-flag

rabl to be transiently transfected was incubated with pl of

Opti-MEM media and 2 pl of Lipofectamine 2000 (Invitrogen,

USA) in a tube at room temperature for 20 minutes, and then

added to each well. Additionally, 450 pl of antibiotic-free

DMEM medium was added to each well which was then incubated

at 37°C in 5% CO 2 for 6 hours, after which the medium was

replaced with 500 pl of 10% FBS-containing DMEM medium,

followed by incubation for 24 hours. Next, each well was

treated with 3 pM of TMab4 in 0.5 ml of fresh medium for 30

minutes, and then washed rapidly three times with PBS and

incubated in medium at 370C for 0, 2 and 6 hours. Thereafter, the medium was removed, and each well was washed with PBS, and then proteins attached to the surface were removed with weakly acidic solution (200 mM glycine, 150 mM NaCl pH 2.5).

After washing with PBS, the cells were fixed in 4%

paraformaldehyde at 250C for 10 minutes.

After washing with PBS, each well was incubated with PBS

buffer containing 0.1% saponin, 0.1% sodium azide and 1% BSA

at 250C for 10 minutes to form pores in the cell membranes.

After washing with PBS, each well was incubated with PBS

buffer containing 2% BSA at 250C for 1 hour to eliminate

nonspecific binding. Then, the cells were stained with an

FITC (green fluorescence) or TRITC (red fluorescence)-labeled

antibody (Sigma) that specifically recognizes human Fc. Rab5

was incubated with anti-rab5 against the early endosome

marker rab5. Each well was incubated with anti-flag

antibodies against a flag-tag of rabl, a recycling endosome

marker, at 250C for 1 hour, and was then incubated with TRITC

(red fluorescence) or FITC (green fluorescence)-labeled

secondary antibody at 250C for 1 hour. To observe late

endosomes and lysosomes, the cells being incubated were

treated with 1 mM LysoTracker Red DND-99 at 30 minutes before

cell fixation. The nucleus was blue-stained with Hoechst33342

and observed with a confocal microscope. As a result, it was

shown that, unlike TAT, TMab4 was located in early endosomes up to 2 hours, and then was not transported to lysosomes or recycling endosomes.

Example 3: Evaluation of the Effect of Acidification in

Early Endosomes on Endosomal Escape

To obtain more clear evidence that the cytosol

penetrating antibody of the present disclosure escapes from

early endosomes, an experiment was performed using inhibitors.

Specifically, the inhibitors used were wortmannin that

inhibits maturation from early endosomes to late endosomes,

bafilomycin that prevents endosomal oxidation by inhibiting

ATPase hydrogen pump, and brefeldin A that inhibits transport

from endosomes to endoplasmic reticulum and Golgi.

FIG. 2a shows the results of confocal microscopy

observation of the cytosol-penetrating ability of the

cytosol-penetrating antibody TMab4 or the cell-penetrating

peptide TAT according to the present disclosure in the

presence or absence of an inhibitor thereof.

Specifically, HeLa cells were prepared in the same

manner as described in Example 2. When the cells were

stabilized, the cells were incubated with each of 100 nM

wortmannin, 200 nM bafilomycin and 7 pM brefeldin A for 30

minutes. Next, the cells were incubated with each of PBS, 2

pM TMab4 and 2 pM TAT at 370C for 6 hours. The cells were

washed with PBS and weakly acidic solution in the same manner as described in Example 2, and then subjected to cell fixation, cell perforation and blocking processes. The TMab4 treated cells were stained with an FITC (green fluorescence) labeled antibody that specifically recognizes human Fc. The nucleus was blue-stained with Hoechst 33342 and observed with a confocal microscope. In the case of TMab4, green fluorescence localized in the cytosol was not observed only in the bafilomycin-treated cells, and spot-shaped fluorescence appeared.

FIG. 2b is a bar graph showing the results of

quantifying the FITC (green fluorescence) fluorescence of the

confocal micrographs shown in FIG. 2a.

Specifically, using Image J software (National

Institutes of Health, USA), 20 cells were selected in each

condition, and then the obtained mean values of fluorescence

are graphically shown.

FIG. 2c shows the results of observing the cytosolic

localization of the cytosol-penetrating antibody TMab4 or the

cell-penetrating peptide TAT according to the present

disclosure by confocal microscopy using calcein in the

presence or absence of an inhibitor thereof.

Specifically, HeLa cells were prepared in the same as

described in Example 2, and were incubated in serum-free

medium with each of 200 nM wortmannin, 200 nM bafilomycin and

7 pM brefeldin A for 30 minutes. Next, the cells were incubated with each of PBS, 2 pM TMab4 and 20 pM TAT at 370C for 6 hours. After 4 hours, each well containing PBS or the antibody was treated with 150 pM calcein and incubated at

370C for 2 hours. In the same manner as described in Example

2, the cells were washed with PBS and weakly acidic solution,

and then fixed.

The nucleus was blue-stained with Hoechst 33342 and

observed with a confocal microscope. As a result, green

calcein fluorescence appeared, indicating that calcein did

escape from endosomes into the cytosol by the cytosol

penetrating antibody TMab4 of the present disclosure and TAT.

However, in the case of TMab4, green calcein fluorescence

localized in the cytosol could not be observed only in the

bafilomycin-treated cells, unlike the cells treated with

other inhibitors.

FIG. 2d is a bar graph showing the results of

quantifying the calcein fluorescence of the confocal

micrographs shown in FIG. 2c.

Specifically, as shown in FIG. 2d, using Image J

software (National Institutes of Health, USA), 20 cells were

selected in each condition, and then the obtained mean values

of fluorescence are graphically shown.

Example 4: Evaluation of the Effect of HSPG Degradation

in Early Endosomes on Endosomal Escape

The cytosol-penetrating antibody is endocytosed by

binding to HSPG on the cell surface. At this time, it is

endocytosed with pro-heparanase. Pro-heparanase is activated

with endosomal acidification (Gingis-Velitski et al., 2004).

Activated heparanase degrades HSPG, and thus the cytosol

penetrating antibody can be freely localized in the cytosol.

FIG. 3a shows the results of Western blot analysis

performed to confirm siRNA (short interfering RNA)-induced

inhibition of heparanase expression.

Specifically, 1 x 104 HeLa cells were added to each well

of 6-well plates and cultured in 1 ml of 10% FBS-containing

medium at 370C in 5% C02 for 12 hours. After 24 hours of

culture, each well was transiently transfected with siRNA.

For transient transfection, 500 ng of each of a control siRNA

having no targeting ability and an siRNA targeting inhibition

of heparanase expression was incubated with 500 pl of Opti

MEM media (Gibco) and 3.5 pl of Lipofectamine 2000

(Invitrogen, USA) in a tube at room temperature for 20

minutes, and then added to each well. 500 pl of antibiotic

free DMEM medium was added to each well which was then

incubated at 370C in 5% C02 for 6 hours. Next, the medium was

preplaced with 1 ml of 10% FBS-containing DMEM medium,

followed by incubation for 72 hours.

After incubation, lysis buffer (10 mM Tris-HCl pH 7.4,

100mM NaCl, 1% SDS, 1mM EDTA, Inhibitor cocktail(sigma)) was added to each well to a cell lysate. The cell lysate was quantified using a BCA protein assay kit (Pierce). The gel subjected to SDS-PAGE was transferred to a PVDF membrane, incubated with the antibody (SantaCruz)(which recognize heparanase and B-actin, respectively) at 250C for 2 hours, and then incubated with HRP-conjugated secondary antibody

(SantaCruz) at 250C for 1 hour, followed by detection.

Analysis was performed using ImageQuant LAS4000 mini (GE

Healthcare).

FIG. 3b shows the results of confocal microscopy

observation of cytosol penetrating antibody/lysosome merging

caused by inhibition of heparanase expression.

Specifically, HeLa cells with inhibited inhibition of

heparanase expression and control HeLa cells were prepared in

the same manner as described in Example 2. The cells were

treated with each of 3 pM TMab4 and 20 pM FITC-TAT at 370C

for 30 minutes, washed rapidly three times with PBS, and then

incubated in medium at 370C for 2 hours. In the same manner

as described in Example 2, the cells were washed with PBS and

weakly acidic solution, and then subjected to cell fixation,

cell perforation and blocking processes.

The TMab4-treated cells were stained with an FITC (green

fluoescence)-labeled antibody that specifically recognizes

human Fc. The cells were incubated with anti-LAMP-1 (santa

cruz) against the lysosome marker LAMP-1 at 250C for 1 hour, and incubated with TRITC (red fluorescence)-labeled secondary antibody at 25°C for 1 hour. The nucleus was blue-stained with Hoechst 33342 and observed with a confocal microscope.

In the case of TMab4, merging with LAMP-1 was observed when

heparanase expression was inhibited.

FIG. 3c shows the results of confocal microscopy

observation performed to confirm the cytosolic localization

of a cytosol-penetrating antibody, which is caused by

inhibition of heparanase expression.

Specifically, HeLa cells with inhibited inhibition of

heparanase expression and control HeLa cells were prepared in

the same manner as described in Example 2. The cells were

treated with each of 2 pM TMab4 and 20 p4M FITC-TAT at 37°C

for 6 hours. After 4 hours, each well containing PBS or the

antibody was treated with 150 pM calcein and incubated at

37°C for 2 hours. In the same manner as described in Example

2, the cells were washed with PBS and weakly acidic solution,

and then fixed. The nucleus was blue-stained with Hoechst

33342 and observed with a confocal microscope. In the cells

with inhibited expression of heparanase, calcein fluorescence

that localized to the cytosol by TMab4 could not be observed.

FIG. 4 is a schematic view showing an overall

trafficking process ranging from cellular internalization of

a cytosol-penetrating antibody according to the present

disclosure to localization of the antibody in the cytosol.

Example 5: Observation of Introduction of Cytosol

Penetrating Intact IgG-Format Monoclonal Antibody through

Cell Membrane at Varying pHs

In order for the cytosol-penetrating antibody of the

present disclosure to localize in the cytosol after

endocytosis, an endosomal escape process is essential. Until

now, there has been no report on endosomal escape of

antibodies. To elucidate the endosomal escape mechanism, an

experiment was performed at simulated endosomal pH.

The components of the inner phospholipid layer of early

endosomes are similar to those of the outer phospholipid

layer of the cell membrane (Bissig and Gruenberg, 2013), and

the major component of the phospholipid layer is 1-palmitoyl

2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Thus,

assuming that the outer phospholipid layer of the membrane of

Ramos cells expressing no HSPG is the same as the inner

phospholipid layer of early endosomes, an experiment was

performed.

FIG. 5 shows the results of observing a fluorescence

labeled cytosol-penetrating antibody in Ramos cells by

confocal microscopy in order to examine whether the antibody

can be introduced through the cell membrane depending on pH

or whether the antibody can induce cell membrane permeation

of other substances.

Specifically, a cover slip was added to 24-well plates,

and 200 pl of 0.01% poly-L-lysine solution was added to

attach suspending Ramos cells to the plate, followed by

incubation at 250C for 20 minutes. After washing with PBS, 5

x 104 Ramos cells were added to each well and incubated in 0.5

ml of 10% FBS-containing medium at 370C for 30 minutes. After

confirming cell adhesion, the cells were incubated in 200 pl

of pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) or pH

5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) with each of 10

pM PBS and TMab4 labeled directly with the fluorescent

reagent DyLight-488, 10 pM non-labeled TMab4 and 2 pM control

antibody adalimumab labeled directly with DyLight-488, at

370C for 2 hours. Adalimumab used as the control antibody is

a therapeutic antibody that targets extracellular cytokines.

In the same manner as described in Example 2, the cells

were washed with PBS, and then fiaxed. The nucleus was blue

stained with Hoechst 33342 and observed with a confocal

microscope. At pH 5.5, the fluorescence of TMab4 labeled

directly with DyLight-488 was observed. At pH 5.5, green

FITC fluorescence was observed in the cells treated with

TMab4 and adalimumab labeled directly with DyLight-488. It

was confirmed that the cytosol-penetrating antibody was

introduced through the cell membrane at acidic pH and could

introduce other substance as well as itself.

In addition, it was confirmed that the morphology of the

cell membrane was maintained, even though the substance was

introduced externally.

Example 6: Examination of Whether Cytosol-Penetrating

Antibody Forms Pores by Trypan Blue Uptake Depending on pH

Among known endosomal escape mechanisms, endosomal

perforation was expected to be the most promising endosomal

escape mechanism by which an intact IgG-format substance can

escape from endosomes while maintaining the morphology of

endosomes as shown in the experimental results.

Similar to Example 5, an experiment was performed in

order to observe the morphology of the cell membrane when the

cytosol-penetrating antibody passed through the cell membrane.

FIG. 6a shows the results of observing Ramos cells by an

optical microscope in order to examine whether a cytosol

penetrating antibody can form pores and take up trypan blue

having no membrane-permeating ability, depending on pH.

pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) or pH

5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5)

Specifically, 5 x 104 Ramos cells were attached to each

well of 24-well plates in the same manner as described in

Example 5. After confirming cell adhesion, the cells were

incubated with each of TMab4 and 1 pM and 10 pM of adalimumab

in 200 pl of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES pH

7.4(cytosol pH)) and pH 5.5 buffer (HBSS(Welgene), 50 mM MES

pH 5.5)(early endosomal pH)) at 370C for 2 hours. After

pl careful washing with PBS, 200 pl of a mixture of 190 of

PBS and 10 pl of trypan blue was added to each well, and the

cells were observed with a microscope.

FIG. 6b is a graph quantitatively comparing the number

of cells that have taken up trypan blue.

Specifically, the number of cells showing trypan blue

uptake was counted and expressed as percentage relative to

the total number of cells. A total of 400 or more cells were

counted, and the mean values are graphically shown.

As shown in FIG. 6b, only at pH 5.5, the cells treated

with the cytosol-penetrating antibody TMab4 of the present

disclosure showed trypan blue uptake in a concentration

dependent manner. In addition, it was shown that the

morphology of the cell membrane during the passage of the

cytosol-penetrating antibody was maintained.

Example 7: Observation of Temporary ad Reversible Pore

Formation by Cytosol-Penetrating Antibody

In the case of conventional peptides known to show a

pore formation mechanism by the endosomal escape mechanism,

it is known that the alpha-helical structure of the peptides

forms pores through the cell membrane.

However, since antibodies have no alpha-helical

structure, they were generally considered almost impossible

to form pores through the cell membrane. Thus, it was assumed

that the antibody would escape from endosomes after temporary

pore formation, and then the cell membrane would be

reversibly restored. To demonstrate this assumption, an

experiment was performed.

FIG 7a shows the results of optical microscopic

observation performed to confirm whether cell membrane pores

produced by a cytosol-penetrating antibody at pH 5.5 is

temporary and reversible.

Specifically, 5 x 104 Ramos cells were attached to each

well of 24-well plates in the same manner as described in

Example 5. After conforming cell adhesion, the cells were

incubated with 10 pM of TMab4 in 200 pl of pH 5.5 buffer

(HBSS (Welgene), 50 mM MES pH 5.5) at 370C for 2 hours in

order to maintain an early endosomal pH of 5.5. The buffer

was replaced with fresh buffer, and the cells were incubated

for 2 hours so that the cells could be recovered. After

careful washing with PBS, 200 pl of a mixture of 190 pl of

PBS and 10 pl of trypan blue was added to each well, and the

cells were observed with a microscope.

FIG. 7b is a graph quantitatively comparing the number

of cells that have taken up trypan blue uptake. Specifically,

a total of 400 or more cells were counted, and the mean values are graphically shown. As shown in FIG. 7b, at pH 5.5, the cells treated with TMab4 having endosomal escape ability according to the present disclosure did take up trypan blue immediately after addition of TMab4, but the cells subjected to recovery in the medium did not take up blue uptake. Namely, it was confirmed that pore formation by the cytosol penetrating antibody was a temporary and reversible phenomenon.

Example 8: Observation of Membrane Binding and Lipid

Membrane Flip-Flop of Cytosol-Penetrating Intact IgG-Format

Monoclonal Antibody at Varying pHs

The pore formation mechanism is a mechanism by which

pores are formed while maintaining the overall morphology of

the cell membrane and a substance escapes from endosomes into

the cytosol through the pores. For pore formation, it is

known that a substance interacts with the inner phospholipid

layer of endosomes, and then membrane pores are formed by a

flip-flop mechanism (H. D. Herce et al., 2009).

Thus, in order for endosomal escape occurs by pore

formation in early endosomes, an antibody should first bind

to the cell membrane by endosomal acidification. To confirm

this, an experiment was performed.

FIG. 8 shows the results of analyzing the cell membrane

binding of a cytosol-penetrating antibody and control

antibody adalimumab by flow cytometry (FACS) at varying pHs.

Specifically, 1x10 5 Ramos were prepared for each sample.

The cells were washed with PBS, and then incubated with each

of 5 pM TMab4 and 5 pM adalimumab in each of pH 7.4 buffer

(TBS, 2% BSA, 50 mM HEPES pH 7.4)(for maintaining a cytosolic

pH of 7.4) and pH 5.5 buffer (TBS, 2% BSA, 50 mM MES pH 5.5)

(for maintaining an early endosomal pH) at 40C for 1 hour.

The cells were washed with each pH buffer, and then the cells

treated with each of TMab4 or adalimumab were incubated with

FITC (green fluorescence)-labeled antibody (which

specifically recognizes human Fc) at 40C for 30 minutes. The

cells were washed with PBS, and then analyzed by flow

cytometry. As a result, it was shown that, at pH 5.5, only

TMab4 did bind to the cell membrane.

FIG. 9 shows the results of analyzing the cell membrane

flip-flop inducing abilities of a cytosol-penetrating antibody

and control antibody adalimumab by flow cytometry (FACS) at

varying pHs.

Specifically, 1x10 5 Ramos cells were prepared for each

sample. The cells were washed with PBS, and then incubated

with each of 5 pM TMab4 and 5 pM adalimumab in each of pH 7.4

buffer (TBS, 2% BSA, 50 mM HEPES pH 7.4) (for maintaining a

cytosolic pH of 7.4) and pH 5.5 buffer (TBS, 2% BSA, 50 mM

MES pH 5.5) (for maintaining an early endosomal pH of 5.5) at

40C for 1 hour.

The cells were washed with each pH buffer, and then

incubated with FITC (green fluorescence)-labeled Annexin-V at

250C for 15 minutes. Annexin-V is a substance that targets

phosphatidylserine, a lipid present only in the cell membrane,

and only when cell membrane lipid flip-flop occurs, the lipid

can be exposed to the outside and Annexin-V can bind thereto.

After washing with PBS, the cells were analyzed by flow

cytometry. As a result, it was confirmed that, at pH 5.5,

Annexin-V did bind only to TMab4.

FIG. 10 is a schematic view showing a pore formation

model of a cytosol-penetrating antibody, expected based on

the above-described experiments.

Example 9: Logic of Prediction of pH-Dependent Change in

Properties

The reason why the cytosol-penetrating antibody

according to the present disclosure showed different cytosol

penetration properties depending on pH was assumed to be

because a pH-dependent change in interaction between antibody

residues led to a change in the properties.

To demonstrate this assumption, literature search was

performed. As a result, it was confirmed that as pH decreases

from 7.4 (neutral pH) to 5.0, aspartic acid (D) and glutamic acid (E) among amino acids lose negative charge by protonation and becomes hydrophobic (Korte et al., 1992).

Specifically, aspartic acid (D) and glutamic acid (E)

, which have become hydrophobic, hydrophobically interact with

methionine (M), leucine (L) and isoleucine (I), which are

originally hydrophobic amino acids. The phenomenon that the

surrounding amino acids induce structural modification

through this newly formed interaction is defined as the

Tanford transition (Qin et al., 1998). To confirm this pH

dependent change in the properties, an experiment was

performed (Di Russo et al., 2012).

In a hT4 VL structure which is a cytosol-penetrating

light-chain variable region, hydrophobic amino acids,

methionine (M), isoleucine (I) and leucine (L), which

surround histidine (H), aspartic acid (D) and glutamic acid

(E), which can show a difference between pH 7.4 and pH 5.0,

were examined.

Among these amino acids, candidate amino acids where the

distance between the side chains of two amino acids was less

than 6-7A were identified, and a pair of the 1st and 9 5 th

amino acids from the N-terminus were selected as candidate

amino acids capable of showing the Tanford transition effect.

Among the pair of the 1st and 9 5 th amino acids, the 9 5 th

amino acid is an amino acid present in the sequence VL-CDR3

of the cytosol-penetrating light-chain variable region hT4 VL.

It was confirmed that the 95th amino acid could induce a

change in the VL-CDR3 loop structure through a phenomenon,

such as the Tanford transition, by interaction with the lst

amino acid.

It was confirmed that, in the cytosol-penetrating light

chain variable region hT4 VL, the amino acids of the VL-CDR3

loop which was structurally changed by the 1st and 9 5 th amino

acids include a very high proportion of tyrosine (Y) which

easily interacts with 1-palmitoyl-2-oleoyl-sn-glycero-3

phosphatidylcholine (POPC) which is the major component of

the inner phospholipid layer of early endosomes (Morita et

al., 2011).

FIG. 11 shows the results of predicting the pH-dependent

structural change of a cytosol-penetrating antibody on the

basis of the WAM modeling structure of the light-chain

variable region of the cytosol-penetrating antibody, and

shows amino acids, which are involved in the structural

change, and amino acids which are exposed by the structural

change.

In order to confirm the pH-dependent change in

properties induced by the 1st and 9 5 th amino acids and the

endosomal escape resulting from the change, mutants were

constructed by substituting the 1st and 9 5 th amino acids with

alanine (A).

In addition, in order to confirm the pH-dependent change

in properties induced by the 1st and 9 5 th amino acids and the

endosomal escape resulting from the change, mutants were

constructed by substituting the 1st and 9 5 th amino acids with

glutamic acid (E) and leucine having properties similar

thereto.

Table 1 shows the names and sequences of mutants

constructed using an overlap PCR technique.

[Table 1]

Name of Variable Sequence SEQ ID NO Region 1 10 20 abcdef 30 40 50 hT4 VL DLVMTOSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQID 60 70 80 90 100 NO:65 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYYHMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 SEQ ID hT4-D1AVL ALVMTOSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW 60 70 80 90 100 NO:66 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYYHMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-M95A VL DLVMTOSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 60 70 80 90 100 NO:67 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYYHAYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-D1E VL ELVMTOSPSSLSASVGDRVTIITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 60 70 80 90 100 NO:68 ASTRESGVPSRFSGSGSGTDFTLTISSLPEDFATYYCQQYYYHMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 SEQ ID hT4-M95L VL DLVMTOSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW 60 70 80 90 100 NO:69 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYYHLYTFGQGTKVEIKR

In the same manner as described in Example 1, cloning,

expression in HEK293F cell lines, and purification were

performed.

Example 10: Observation of pH-Dependent Change in

Properties of Cytosol-Penetrating Antibody

FIG. 12 is a graph quantitatively comparing the number

of cells that have taken up trypan blue at varying pHs by

mutants (TMab4-D1A), (TMab4-M95A), (TMab4-D1E), and (TMab4

M95L) constructed by substituting the 1st amino acid aspartic

acid (D), the 9 5 th amino acid methionine (M), the 1st amino

acid aspartic acid (A), and the 9 5 th amino acid methionine (M)

of a light-chain variable region (VL), which are involved in

induction of a structural change of a cytosol-penetrating

antibody at acidic pH, with alanine (A), alanine (A),

glutamic acid (E), and leucine (L), respectively.

Specifically, Ramos cells were attached to plates in the

same manner as described in Example 5. Then, the cells were

incubated with lOpM of each of TMab4, Adalimumab, TMab4-D1A,

TMab4-M95A, TMab4-D1E and TMab4-M95L in 200 pl of each of pH

7.4 buffer (HBSS(Welgene), 50 mM HEPES pH 7.4)(for

maintaining a cytosolic pH of 7.4) and pH 5.5 buffer

(HBSS(Welgene), 50 mM MES pH 5.5) (for maintaining an early

endosomal pH of 5.5) at 370C for 2 hours.

After careful washing with PBS, 200 pl of a mixture of

190 pl of PBS and 10 pl of trypan blue was added to each well,

and the cells were observed with a microscope. The number of

cells showing trypan blue uptake was counted and expressed as

percentage relative to the total number of cells. A total of

400 or more cells were counted, and the mean values are

graphically shown.

It was confirmed that the mutants, TMab4-D1A and TMab4

M95A, showed little or no trypan blue uptake, unlike TMab4.

TMab4-D1E and TMab4-M95L showed trypan blue uptake similar to

that of TMab4. This suggests that the 1 st amino acid and the

95th amino acid play an important role in endosomal escape.

Example 11: Investigation of Amino Acids and Motifs

Contributing to Endosomal Escape Ability of Cytosol

Penetrating Antibody

Through the experimental examples obtained in the above

Examples, it was found that the pH-dependent change in the

properties of the antibody occurred by interaction with the

1 't and 95th antibodies of the cytosol-penetrating antibody and

that endosomal escape was induced by the change in the

properties.

In order to confirm endosomal escape induced by the pH

dependent change in the properties, mutants were constructed by substituting amino acids of VL-CDR3, which were expected to interact with phospholipid, with alanine (A).

Specifically, based on the results of structural

modeling analysis, mutants were constructed by simultaneously

substituting the 9 2 "d, 9 3 rd and 9 4 th amino acids, which were

most likely to be exposed to the surface, with alanine (A).

Table 2 below shows the names and sequences of mutants

constructed using an overlap PCR technique.

[Table 2]

Name of Variable Sequence SEQ ID NO: Region 1 10 20 abcdef 30 40 50 hT4-Y91A VL DLVMTOSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 60 70 80 90 100 NO:70 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYYHMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 SEQ ID hT4-Y92A VL DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQOKPGKAPKLLIYW 60 70 80 90 100 NO:71 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYAYHMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-Y93A VL DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 60 70 80 90 100 NO:72 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAHMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-H94A VL DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 60 70 80 90 100 NO:73 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYYAMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-AAA VL DLVMTOSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 60 70 80 90 100 NO:74 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYAAAMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 SEQ ID hT4-Y96A VL DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW 60 70 80 90 100 NO:75 ASTRESGVPSRFSGSGSGTDFTLTISSLPEDFATYYCQQYYYHMATFGQGTKVEIKR

In the same manner as described in Example 1, cloning,

expression in HEK293F cell lines, and purification were

performed.

FIG. 13 is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by

mutants constructed by substituting the 9 2nd, 93 , and 9 4 th

amino acids of the CDR3 of the light-chain variable region

(VL) of a cytosol-penetrating antibody, which can possibly be

involved in endosomal escape, with alanine.

Specifically, Ramos cells were attached to plates in the

same manner as described in Example 5. Then, the cells were

incubated with each of buffer and 10 pM of TMab4, TMab4-Y91A,

TMab4-Y92A, TMab4-Y93A, TMab4-H94A, TMab4-AAA and TMab4-Y96A

in 200 g0 of each of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES

pH 7.4)(for maintaining a cytosolic pH of 7.4) and pH 5.5

buffer (HBSS(Welgene), 50 mM MES pH 5.5)(for maintaining an

early endosomal pH of 5.5) at 370C for 2 hours.

After careful washing with PBS, 200 pl of a mixture of

190 pl of PBS and 10 pl of trypan blue was added to each well,

and the cells were observed with a microscope. The number of

cells showing trypan blue uptake was counted and expressed as

percentage relative to the total number of cells. A total of

400 or more cells were counted, and the mean values are

graphically shown. It was shown that TMab4-Y92A, TMab4-Y93A

and TMab4-H94A showed significantly reduced trypan blue uptake compared to TMab4. In particular, TMab4-AAA showed little or no trypan blue uptake. However, TMab4-Y91A and

TMab4-Y96A showed trypan blue uptake similar to that of TMab4.

This suggests that the 9 2nd, 9 3r and 9 4 th amino acids greatly

contribute to endosomal escape.

Example 12: Confirmation of Contribution of CDR1 and

CDR2 of Cytosol-Penetrating Antibody Light-Chain Variable

Region (VL) to Endosomal Escape

The above-described experimental results demonstrated

that the CDR3 of the light-chain variable region (VL) is

involved in endosomal escape. Then, in order to elucidate the

effect of the CDR1 and CDR2 of the light-chain variable

region (VL), which are involved in endocytosis, on endosomal

escape, an experiment was performed.

The CDR1 and CDR2 of the light-chain variable region

(VL) were substituted with CDR sequences which have the same

amino acid number or do not include the cationic patch

sequence of CDR1 involved in endocytosis, among human

germline sequences. At this time, amino acids known to be

important for the stability of the existing light-chain

variable region were conserved.

Table 3 below shows the names and sequences of mutants

constructed using genetic synthesis.

[Table 3]

Name of Variable Sequence SEQ ID NO: Region 1 10 20 abcd 40 50 SEQ ID hT4-01 VL DLVMTOSPSSLSASVGDRVTITCKASOGLSSYLAWYQQKPGKAPKLLIYW 60 70 80 90 100 NO:76 ASTLESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQYYYHMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 SEQ ID hT4-02 VL DLVMTOSPSSLSASVGDRVTITCKSSOSLLYSSNNKNYLAWYOQKPGKAPKLLIYW 60 70 80 90 100 N0:77 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQYYYHMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 SEQ 1D hT4-03 VL DLVMTOSPSSLSASVGDRVTITCKSSOSLLDSDDGNTYLAWYOKPGKAPKLLIYW 60 70 80 90 100 NO:78 LSYRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYYHMYTFGQGTKVEIKR

In the same manner as described in Example 1, cloning,

expression in HEK293F cell lines, and purification were

performed.

FIG. 14a shows the results of confocal microscopy

performed to analyze the cytosol-penetrating ability of

mutants constructed by substituting the CDR1 and CDR2 of the

light-chain variable region (VL) of a cytosol-penetrating

antibody, which bind to HSPG receptor and are involved in

cytosol-penetrating ability, with human germline sequences.

Specifically, HeLa cells were prepared in the same

manner as described in Example 2. When the cells were

stabilized, the cells were incubated with each of PBS and 2

pM TMab4, TMab4-01, TMab4-02 and TMab4-03 at 37°C for 6 hours.

The cells were washed with PBS and weakly acidic solution in the same manner as described in Example 2, and then subjected to cell fixation, cell perforation and blocking processes.

TMab4 was stained with an Alexa-488 (green

fluorescence)-labeled antibody that specifically recognizes

human Fc. The nucleus was blue-stained with Hoechst33342 and

observed with a confocal microscope. All the three mutants

showed reduced intracellular fluorescence compared to wild

type TMab4. In particular, in the case of TMab4-03, little or

no intracellular fluorescence was observed.

FIG. 14b shows a graph quantitatively comparing the

number of cells that have taken up trypan blue depending on pH

by mutants constructed by substituting the CDR1 and CDR2 of

the light-chain variable region (VL) of a cytosol-penetrating

antibody, which bind to HSPG receptor and are involved in

cytosol-penetrating ability, with human germline sequences.

Specifically, Ramos cells were attached to plates in the

same manner as described in Example 5. Then, the cells were

incubated with each of buffer and 10 pM of TMab4, TMab4-01,

TMab4-02 and TMab4-03 in 200 #0 of each of pH 7.4 buffer

(HBSS(Welgene), 50 mM HEPES pH 7.4)(for maintaining a

cytosolic pH of 7.4) and pH 5.5 buffer (HBSS(Welgene), 50 mM

MES pH 5.5)(for maintaining an early endosomal pH of 5.5) at

370C for 2 hours. After careful washing with PBS, 200 pl of a

mixture of 190 pl of PBS and 10 pl of trypan blue was added

to each well, and the cells were observed with a microscope.

The number of cells showing trypan blue uptake was counted

and expressed as percentage relative to the total number of

cells. A total of 400 or more cells were counted, and the

mean values are graphically shown. As a result, the mutants,

TMab4-01 and TMab4-03, showed trypan blue uptake similar to

that of TMab4. Namely, it was demonstrated that, in the

light-chain variable region, the region involved in

endocytosis (VL-CDR1 and VL-CDR2) is distinguished from the

region involved in endosomal escape (VL-CDR3).

Example 13: Logic of Improvement in Endosomal Escape

Ability of Cytosol-Penetrating Antibody

The 9 2n, 9 3rd and 9 4 th amino acids are expected to

increase solvent accessibility for binding to the inner

phospholipid membrane of early endosomes, which is the early

mechanism of endosomal escape, through the change in

properties of VL-CDR3 by interaction with the 1 st and 9 5 th

amino acids of the cytosol-penetrating light-chain variable

region. These amino acids are tyrosine (Y), tyrosine (Y) and

histidine (H), respectively.

These amino acids easily interact with 1-palmitoyl-2

oleoyl-sn-glycero-3-phosphatidylcholine (POPC) which is the

major component of the inner phospholipid layer of early

endosomes.

In order to confirm that the three amino acids expected

to be exposed due to a change in pH conditions interact with

the inner phospholipid layer of early endosomes and are

involved in endosomal escape and to increase the proportion

of cytosol-penetrating antibody that escapes from endosomes,

mutants for the 9 2nd 9 3 rd and 9 4 th amino acids were

constructed.

For mutant construction, literature search was performed,

and as a result, amino acids that easily interact with 1

palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)

were selected (Morita et al., 2011). The mutant design was

made such that the selected amino acids are introduced into

the 9 2n, 9 3 rd and 9 4 th amino acids.

Specifically, the average binding activity of 20

different amino acids for 1-palmitoyl-2-oleoyl-sn-glycero-3

phosphatidylcholine (POPC) is higher in the order of

tryptophan (W), phenylalanine (F), tyrosine (Y), leucine (L),

isoleucine (I), cysteine (C), and methionine (M).

Specifically, the binding activity of 20 different amino

acids for the hydrophilic head portion of 1-palmitoyl-2

oleoyl-sn-glycero-3-phosphatidylcholine (POPC) is higher in

the order of arginine (R), tryptophan (W), tyrosine (Y),

histidine (H), asparagine (N), glutamine (Q), lysine (K),

and phenylalanine (F) . In addition, the binding activity of

20 different amino acids for the hydrophobic head portion of

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)

is higher in the order of tryptophan (W), phenylalanine (F),

leucine (L) , methionine (M) , isoleucine (I) , valine V) , and

tyrosine (Y).

Based on such results, it was confirmed that tryptophan

(W) is an amino acid that most easily interacts with POPC

which is the major component of the inner phospholipid layer

of early endosomes (Morita et al., 2011). Thus, in the

present disclosure, a strategy of substituting one or two

amino acids with tryptophan (W) was adopted.

Tables 4, 5 and 6 below show the sequences of the

designed mutant light-chain variable regions expected to

improve the endosomal escape ability of the human antibody

having cytosol-penetrating ability. Table 4 below shows the

full-length sequences of the light-chain variable regions of

the human antibody according to the Kabat numbering system,

and Tables 5 and 6 below show the CDR1 and CDR2 sequences or

CDR3 sequences of the antibody sequences shown in Table 4.

[Table 4]

Name of Variable Sequence SEQ ID NO: Region 1 10 20 abcdef 30 40 50 SEQ ID hT4-WWH VL DLVMTOSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYQQKPGKAPKLLIYW 60 70 80 90 100 NO:1 ASTRESGVPSRFSGSGSGTDFTLTISSL0PEDFATYYCQQYWWHMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-WYW VL DLVMTOSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 60 70 80 90 100 NO:2 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQYWYWMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-YWWVL DLVMTOSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 60 70 80 90 100 NO:3 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQYYWWMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-WYH VL DLVMTOSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 60 70 80 90 100 NO:4 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQYWYHMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-YWH VL DLVMTOSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 60 70 80 90 100 NO:5 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQYYWHMYTFGQGTKVEIKR

[Table 5]

CDR2 CDR1 Sequence Sequence

KaSbatN.n Light Chain Variable K S S Q S L F N S R T R K N Y L A W A S T R E S Region

SEQ ID NO: SEQ ID NO:6 SEQ ID NO:7

[Table 6]

Name of Light SEQID Chain CDR3 Sequence NO: Variable Region Kabat No.

SEQ ID hT4-WWH VL QQYWWHMYT MY NO:8 SEQ ID hT4-WYW VL QQYWYWMYT NO:9 SEQ ID hT4-YWW VL QQYYWWMYT MY NO:10 SEQ ID hT4-WYH VL QQYWYHMYT MY NO:11 SEQ ID hT4-YWHVL QQYYWHMYT MY NO:12 Example 14: Expression and Purification of Cytosol

Penetrating Antibody Mutants Expected to Have Increased

Endosomal Escape Ability and Confirmation of Maintenance of

Cytosol-Penetrating Ability

For animal cell expression of cytosol-penetrating

antibody mutants expected to have increased endosomal escape

ability, vectors expressing the light chain were constructed

as described in Example 1 above. To this end, DNA encoding a

light chain comprising the cytosol-penetrating light-chain variable region (hT4 VL) or the mutant antibody's light-chain variable region (hT4-WWH VL, hT4-WYW VL, hT4-YWW VL, hT4-WYH

VL, hT4-YWH VL) and light chain constant region (CL), which

has a secretion signal peptide-encoding DNA fused to the 5'

end, was cloned into a pcDNA3.4 vector (Invitrogen) by use of

NotI/HindIII.

Next, a humanized hTO VH-encoding animal expression

vector and the constructed animal expression vector encoding

the light chain comprising the light-chain variable region

expected to have increased endosomal escape ability were

transiently transfected into HEK293F protein-expressing cells.

Next, purification of the cytosol-penetrating antibody mutant

expected to have increased endosomal escape ability was

performed in the same manner as described in Example 1.

FIG. 15a shows the results of 12% SDS-PAGE analysis

under reducing or non-reducing conditions after purification

of cytosol-penetrating antibody mutants expected to have

improved endosomal escape ability.

Specifically, under non-reducing conditions, a molecular

weight of about 150 kDa was observed, and under reducing

conditions, the heavy chain showed a molecular weight of 50

kDa, and the light chain showed a molecular weight of 25 kDa.

This suggests that the expression and purified cytosol

penetrating antibody mutants expected to have increased

endosomal escape ability are present as monomers in a solution state and do not form dimers or oligomers through non-natural disulfide bonds.

FIG. 15b shows the results of confocal microscopy

performed to examine whether the cytosol-penetrating ability

of cytosol-penetrating antibody mutants expected to have

improved endosomal escape ability is maintained.

Specifically, HeLa cells were prepared in the same

manner as described in Example 2 above. When the cells were

stabilized, the cells were incubated with each of PBS and 2

pM TMab4, TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and

TMab4-YWH at 370C for 6 hours.

The cells were washed with PBS and weakly acidic

solution in the same manner as described in Example 2, and

then subjected to cell fixation, cell perforation and

blocking processes. Each antibody was stained with an FITC

(green fluorescence)-labeled antibody that specifically

recognizes human Fc. It was found that in all the five

mutants, the cytosol-penetrating ability was maintained.

Example 15: Confirmation of pH Dependence of Cytosol

Penetrating Antibody Mutants Expected to Have Increased

Endosomal Escape Ability

FIG. 16 is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by a

cytosol-penetrating antibody wild-type and cytosol penetrating antibody mutants expected to have improved endosomal escape ability.

Specifically, Ramos cells were attached to plates in the

same manner as described in Example 5. Then, the cells were

incubated with each of 1 pM TMab4, Adalimumab, TMab4-WWH,

TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH in 200 #0 of

each of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES pH

7.4(cytosol pH)) and pH 5.5 buffer (HBSS(Welgene), 50 mM MES

pH 5.5(early endosomal pH)) (early endosomal pH) at 370C for

2 hours. After careful washing with PBS, 200 pl of a mixture

of 190 pl of PBS and 10 pl of trypan blue was added to each

well, and the cells were observed with a microscope. The

number of cells showing trypan blue uptake was counted and

expressed as percentage relative to the total number of cells.

A total of 400 or more cells were counted, and the mean

values are graphically shown. Among the five mutants, TMab4

WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH showed increased

trypan blue uptake, and among them, TMab4-WYW showed pH

dependent trypan blue uptake.

TMab4-WYW, which showed increased pH-dependent trypan

blue uptake while retaining the cytosol-penetrating ability

of the wild-type antibody, was selected as a final clone.

Example 16: Confirmation of Improvement in Cytosol

Localization of Cytosol-Penetrating Antibody Mutant Having

Increased Endosomal Escape Ability

FIG. 17a shows the results of observing the cytosolic

localization of a cytosol-penetrating antibody wild-type and

cytosol-penetrating antibody mutants expected to have

improved endosomal escape ability, by confocal microscopy

using calcein.

Specifically, HeLa cells were prepared in the same

manner as described in Example 2. The cells were incubated

with PBS or 0.1 pM, 0.5 pM and 1 pM of each of TMab4 and

TMab4-WYW in serum-free medium at 370C for 6 hours. After 4

hours, each well containing PBS or the antibody was treated

with 150 pM calcein and incubated at 370C for 2 hours. The

cells were washed with PBS and weakly acidic solution in the

same manner as described in Example 2, and then fixed. The

nucleus was blue-stained with Hoechst33342 and observed with

a confocal microscope. It was confirmed that TMab4-WYW showed

green calcein fluorescence with higher intensity even at

lower concentration than TMab4.

FIG. 17b is a bar graph showing the results of

quantifying the calcein fluorescence of the confocal

micrographs shown in FIG. 17a.

Specifically, using Image J software (National

Institutes of Health, USA), 20 cells were selected in each condition, and then the obtained mean values of fluorescence are graphically shown.

Example 17: Confirmation of Cytosol Localization of

Cytosol-Penetrating Monoclonal Antibody by Enhanced Split-GFP

Complementation Assay

FIG. 18 is a schematic view showing a process in which

GFP fluorescence by enhanced split-GFP complementation is

observed when a cytosol-penetrating antibody wild-type and a

mutant having improved endosomal escape ability localizes in

the cytosol.

Specifically, an enhanced split-GFP complementation

system was used to confirm that the cytosol-penetrating

antibody would localize to the cytosol. When the green

fluorescence protein GFP is split into a fragment 1-10 and a

fragment 11, the fluorescent property is removed, and when

the two fragments become closer to each other and are

combined with each other, the fluorescent property can be

restored (Cabantous et al., 2005).

Based on this property, the GFP fragment 1-10 was

expressed in the cytosol, and the GFP fragment 11 was fused

to the C-terminus of the cytosol-penetrating antibody. In

addition, for complementation between the GFP fragments,

streptavidin and streptavidin-binding peptide 2 (SBP2) having

high affinity were fused to the GFP fragments. Thus, the fact that GFP fluorescence indicates that the cytosol-penetrating antibody localizes in the cytosol.

Example 18: Expression and Purification of Cytosol

Penetrating Antibody Fused with GFP11-SBP2

For expression of a GFP11-SBP2-fused cytosol-penetrating

antibody in animal cells, GFP11-SBP2 was genetically fused to

the C-terminus of the heavy chain by three GGGGS linkers.

Next, the animal expression vector encoding the cytosol

penetrating light chain or the cytosol-penetrating light

chain having increased endosomal escape ability, and the

animal expression vector encoding the GFP11-SBP2-fused heavy

chain, were transiently co-transfected. Next, purification of

the GFP11-SBP2-fused cytosol-penetrating antibody was

performed in the same manner as described in Example 1.

FIG. 19 shows the results of 12% SDS-PAGE analysis under

reducing or non-reducing conditions after purification of a

GFP11-SBP2-fused cytosol-penetrating antibody wild-type and a

GFP11-SBP2-fused mutant having improved endosomal escape

ability.

Specifically, under non-reducing conditions, a molecular

weight of about 150 kDa was observed, and under reducing

conditions, the heavy chain showed a molecular weight of 50

kDa, and the light chain showed a molecular weight of 25 kDa.

This suggests that the expressed purified GFP11-SBP2-fused cytosol-penetrating antibody is present as a monomer in a solution state and does not form a dimer or an oligomer by a non-natural disulfide bond.

Example 19: Examination of GFP Fluorescence with Cytosol

Localization of GFP11-SBP2-Fused Cytosol-Penetrating Antibody

FIG. 20a shows the results of confocal microscopy

performed to examine the GFP fluorescence of a GFP11-SBP2

fused cytosol-penetrating antibody wild-type and a GFP11

SBP2-fused mutant having improved endosomal escape ability by

enhanced split-GFP complementation.

Specifically, transformed HeLa cells stably expressing

SA-GFP1-10 were prepared in the same manner as described in

Example 2. When the cells were stabilized, the cells were

incubated with PBS or 0.2, 0.4, 0.6, 0.8, 1.6 or 3.2 pM of

each of TMab4-GFP11-SBP2 and TMab4-WYW-GFP11-SBP2 at 370C for

6 hours.

In the same manner as described in Example 2, the cells

were washed with PBS and weakly acidic solution, and then

fixed. The nucleus was blue-stained with Hoechst 33342 and

observed with a confocal microscope. It was observed that

TMab4-WYW showed green GFP fluorescence with higher intensity

at lower concentration than TMab4.

FIG. 20b is a graph showing the results of quantifying

the GFP fluorescence of the confocal micrographs shown in FIG.

20a.

Specifically, using Image J software (National

Institutes of Health, USA), 20 cells were selected in each

condition, and then the obtained mean values of fluorescence

are graphically shown.

In order to quantitatively express and compare the

intracytosolic concentrations and endosomal escape

efficiencies of the GFP11-SBP2-fused intact IgG-format

antibody and the cytosol-penetrating antibody having

increased endosomal escape ability, an experiment was

performed.

Table 7 below shows the intracytosolic concentrations

and endosomal escape efficiencies of the GFP11-SBP2-fused

intact IgG-format antibody and the cytosol-penetrating

antibody having increased endosomal escape ability.

[Table 7]

Treated concentrations (pM)' Parameters. cytetransmabs. 0.14' 0.54' 10

Cytosolic concentration TMab4' 12t 54' 68± 4- 170± 9P TMab4-WYW' 34t 7P 23299' 527 t 35P

Endosomal escaping efficiency TMab4P 1.0.41 2.6 t 0.14 4.3+0.1' TMab4-WYW' 3.2 0.4- 8.7 t 0.1o 13.2 ± 0.5o

Example 20: In-Depth Analysis of Interaction between

Cytosol-Penetrating Antibody Having Increased Endosomal

Escape Ability and Lipid

It was found that when the 9 2 "d and 9 4 th amino acids of

the light-chain variable region CDR3 of the wild-type

cytosol-penetrating antibody were substituted with tryptophan,

the endosomal escape ability was increased.

In order to determine whether this increase in the

endosomal escape ability is due to improved interaction with

any part of the lipid, an experiment was performed.

Tryptophan (W) is an amino acid that easily interacts with

both the hydrophilic head and hydrophobic tail of the lipid.

When tryptophan is substituted with arginine (R) (which

easily interacts with the hydrophilic head), isoleucine (I)

(which easily interacts with the hydrophilic tail) or glycine

(G) (which very weakly interacts with the lipid) and the

activities are compared, it can be seen that the interaction

with any part of the lipid plays an important role.

In order to analyze in depth the interaction between the

cytosol-penetrating antibody having increased endosomal

escape ability and the lipid, mutants were constructed by

substituting tryptophan with each of arginine (R), isoleucine

(I) and glycine (G).

Table 8 below shows the names and sequences of the

mutants constructed using an overlap PCR technique.

[Table 8]

Name of Variable Sequence SEQ ID NO: Region 1 10 20 abcdef 30 40 50 SEQ ID hT4-RYR VL DLVMTOSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW 60 70 80 90 100 NO:13 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRYRMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 SEQ ID hT4-IYl VL DLVMTOSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW 60 70 80 90 100 NO:14 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYIYIMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 DLVMTOSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID hT4-GYGVL 60 70 80 90 100 NO:15 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYGYGMYTFGQGTKVEIKR

In the same manner as described in Example 1, cloning,

expression in HEK293F cell lines, and purification were

performed.

Example 21: In-Depth Analysis of Interaction between

Cytosol-Penetrating Antibody and Lipid

FIG. 21a is a graph showing the results of flow

cytometry (FACS) performed to analyze the cell membrane

binding of mutants obtained by substitution with arginine,

isoleucine and glycine, which are amino acids having

properties opposite to those of tryptophan.

Specifically, 1 x 105 Ramos cells were prepared for each

well. The cells were washed with PBS, and then incubated with

each of 3 pM TMab4, TMab4-WYW, TMab4-RYR, TMab4-IYI and

TMab4-GYG in each of pH 7.4 buffer (TBS, 2% BSA, 50 mM HEPES

pH 7.4(cytosolic pH)), and pH 5.5 buffer (TBS, 2% BSA, 50 mM

MES pH 5.5(early endosomal pH)) at 40C for 1 hour. After

washing with each pH buffer, TMab4, TMab4-WYW, TMab4-RYR,

TMab4-IYI and TMab4-GYG were incubated with an FITC (green

fluorescence)-labeled antibody (which specifically recognizes

human Fc) at 40C for 30 minutes. After washing with PBS, the

cells were analyzed by flow cytometry, and as a result, it

was found that, at pH 5.5, only TMab4 did bind to the cell

membrane.

FIG. 21b is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by

mutants obtained by substitution with arginine, isoleucine

and glycine, which are amino acids having properties opposite

to those of tryptophan.

Specifically, Ramos cells were attached to plates in the

same manner as described in Example 5. Then, the cells were

incubated with each of 1 pM TMab4, TMab4-WYW, TMab4-RYR,

TMab4-IYI and TMab4-GYG in 200 pl of each of pH 7.4 buffer

(HBSS(Welgene), 50 mM HEPES pH 7.4 (cytosolic pH)) and pH 5.5

buffer (HBSS(Welgene), 50 mM MES pH 5.5(early endosomal pH))

at 40C for 2 hours. After careful washing with PBS, 200 pl of

a mixture of 190 pl of PBS and 10 pl of trypan blue was added

to each well, and the cells were observed with a microscope.

The number of cells showing trypan blue uptake was counted and expressed as percentage relative to the total number of cells. A total of 400 or more cells were counted, and the mean values are graphically shown. As a result, TMab4-RYR,

TMab4-IYI and TMab4-GYG showed reduced trypan blue uptake

compared to TMab4-WYW.

FIG. 21c is a bar graph showing the results of observing

the cytosolic localization of mutants obtained by

substitution with arginine, isoleucine and glycine, which are

amino acids having properties opposite to those of tryptophan

by confocal microscopy using calcein and quantifying the

calcein fluorescence of the confocal micrographs.

Specifically, HeLa cells were prepared in the same

manner as described in Example 2. The cells were incubated

with each of 0.5 pM TMab4, TMab4-WYW, TMab4-RYR, TMab4-IYI

and TMab4-GYG 0.5 pM at 370C for 6 hours. After 4 hours, each

well containing PBS or the antibody was treated with 150 pM

calcein 150 pM and incubated at 370C for 2 hours. In the same

manner as described in Example 2, the cells were washed with

PBS and weakly acidic solution, and then fixed. The nucleus

was blue-stained with Hoechst33342 and observed with a

confocal microscope. In the cells treated with TMab4-RYR,

TMab4-IYI or TMab4-GYG, the green calcein fluorescence

localized in the cytosol was weaker than that in the cells

treated with TMab4-WYW.

Therefore, it was confirmed that interactions with all

the hydrophilic head and hydrophobic tail of the lipid were

involved in endosomal escape, and for this reason,

substitution with tryptophan increased the endosomal escape

ability.

Example 22: Expression and Purification of Intact IgG

Format Anti-Tubulin Cytosol-Penetrating Antibody

The mutant having increased endosomal escape ability can

more effectively target a protein located in the cytosol,

because the amount of antibody located in the cytosol will

increase.

FIG. 22a is a schematic view showing a process of

constructing an intact IgG-format anti-tubulin cytosol

penetrating antibody to be used to examine the activity of

cytosol-penetrating antibody mutants having improved

endosomal escape ability.

For the purpose of expression of an intact IgG-format

anti-tubulin cytosol-penetrating antibody in animal cells,

DNA encoding a heavy chain comprising the heavy-chain

variable region and heavy chain constant region (CH1-hinge

CH2-CH3) binding specifically to cytoskeletal tubulin, which

has a secretion signal peptide-encoding DNA fused to the 5'

end, was cloned into a pcDNA3.4 vector (Invitrogen) by use of

NotI/HindIII (Laurence et al., 2011).

Next, the animal expression vector encoding the cytosol

penetrating light chain or the cytosol-penetrating light

chain having increased endosomal escape ability, and the

constructed animal expression vector encoding the heavy chain

comprising the heavy-chain variable region that specifically

to tubulin, were transiently co-transfected into HEK293F

protein-expressing cells. Next, purification of the intact

IgG-format anti-tubulin cytosol-penetrating antibody was

performed in the same manner as described in Example 1.

FIG. 22b shows the results of 12% SDS-PAGE analysis

under reducing or non-reducing conditions after purification

of an intact IgG-format anti-tubulin cytosol-penetrating

antibody.

Specifically, under non-reducing conditions, a molecular

weight of about 150 kDa was observed, and under reducing

conditions, the heavy chain showed a molecular weight of 50

kDa, and the light chain showed a molecular weight of 25 kDa.

This suggests that the expressed purified intact IgG-format

anti-tubulin cytosol-penetrating antibody is present as a

monomer in a solution state and does not form a dimer or an

oligomer by a non-natural disulfide bond.

Example 23: Confirmation of Cytoskeletal Tubulin

Specific Binding of Intact IgG-Format Anti-Tubulin Cytosol

Penetrating Antibody

FIG. 22c shows the results of confocal microscopy

performed to examine whether an intact IgG-format anti

tubulin cytosol-penetrating antibody would merge with

cytoskeletal tubulin localized in the cytosol.

Specifically, HeLa cells were prepared in the same

manner as described in Example 2. The cells were incubated

with PBS or each of 3 pM TuT4 and TuT4-WYW in 500 pl of 10%

FBS-containing medium at 370C for 6 hours. The cells were

washed with PBS and weakly acidic solution in the same manner

as described in Example 2, and then subjected to cell

fixation, cell perforation and blocking processes.

Cytoskeletal tubulin was incubated with anti-tubulin

antibody (Santa Cruz) at 250C for 1 hour, and incubated with

TRITC (red fluorescence)-labeled secondary antibody at 250C

for 1 hour. Each antibody was stained with an FITC (green

fluorescence)-labeled antibody that specifically recognizes

human Fc. The nucleus was blue-stained with Hoechst33342 and

observed with a confocal microscope.

As shown in FIG. 22c, with the cytosol portion in which

red fluorescent tubulin was localized, green fluorescent

TuT4-WYW was merged in a fibrillar shape, but TuT4 was not

merged.

Example 24: Expression and Purification of Intact IgG

Format RAS-Targeting Cell-Penetrating Antibody and Analysis

of Affinities of K-RAS Mutants

In order to confirm whether the cytosol-penetrating

antibody can effectively target other intracytosolic proteins

in addition to cytoskeletal tubulin, an experiment was

performed.

FIG. 23a is a schematic view showing a process of

constructing an intact IgG-format RAS-targeting cytosol

penetrating antibody to be used to examine the activity of

mutants having improved endosomal escape ability.

For the purpose of expression of an intact IgG-format

Ras-targeting cytosol-penetrating antibody in animal cells,

DNA encoding a heavy chain the heavy-chain variable region

(RT11 VH) and heavy chain constant region (CH1-hinge-CH2-CH3)

binding specifically to GTP-bound K-RAS, which has a

secretion signal-encoding DNA fused to the 5' end, was cloned

into a pcDNA3.4 vector (Invitrogen) by use of NotI/HindIII as

described in Example 5.

Next, the animal expression vector encoding the cytosol

penetrating light chain or the cytosol-penetrating light

chain having increased endosomal escape ability, and the

constructed animal expression vector encoding the heavy chain

comprising the heavy-chain variable region that binds

specifically to GTP-bound K-RAS, were transiently co transfected into HEK293F protein-expressing cells. Next, purification of the intact IgG-format Ras-targeting cytosol penetrating antibody was performed in the same manner as described above.

FIG. 23b shows the results of 12% SDS-PAGE analysis

under reducing or non-reducing conditions after purification

of intact IgG-format RAS-targeting cytosol-penetrating

antibodies.

Specifically, under non-reducing conditions, a molecular

weight of about 150 kDa was observed, and under reducing

conditions, the heavy chain showed a molecular weight of 50

kDa, and the light chain showed a molecular weight of 25 kDa.

This suggests that the expressed purified intact IgG-format

Ras-targeting cytosol-penetrating antibody is present as a

monomer in a solution state and does not form a dimer or an

oligomer by a non-natural disulfide bond.

FIG. 23c shows the results of enzyme linked

immunosorbent assay performed to measure the affinities of

antibodies for GppNHp-bound K-RAS G12D and GDP-bound K-RAS

G12D, which are K-RAS mutants.

Specifically, GTP is very easily hydrolyzed, and hence

it is difficult to maintain the morphology of GTP-bound K-RAS

G12D. Thus, in order to enable K-RAS to have an activated

structure, like GTP, a GTP-bound K-RAS G12D antigen was

constructed using GppNHp which is a non-hydrolyzable GTP analogue. Each of a GppNHp-bound K-RAS G12D and a GDP-bound

K-RAS G12D, which are target molecules, was incubated in 96

well EIA/RIA plates (COSTAR Corning) at 370C for 1 hour,

followed by washing three times with 0.1% PBST (0.1

% Tween20, pH 7.4, 137 mM NaCl, 12mM phosphate, 2.7 mM KCl)

(SIGMA) for 10 minutes each time. Each well was incubated

with 5% PBSS (5% Skim milk, pH7.4, 137 mM NaCl, 12mM

phosphate, 2.7 mM KCl) (SIGMA) for 1 hour, and then washed

three times with 0.1% PBST for 10 minutes. Next, each well

was incubated with each of the IgG-format cytosol-penetrating

antibodies (TMab4, RT11, and RT11-WYW), and then washed three

times with 0.1% PBST for 10 minutes. As a marker antibody,

goat alkaline phosphatase-conjugated anti-human mAb (SIGMA)

was used. Each well was treated with pNPP (p-nitrophenyl

palmitate) (Sigma), and the absorbance at 405 nm was measured.

Affinities for the K-RAS mutants were analyzed, and as a

result, it was shown that there was little or no difference

in affinity between wild-type RT11 and mutant RT11-WYW. TMab4

used as a negative control did not bind, and all the clones

did not bind to the GDP-bound K-RASs.

Example 25: Confirmation of Specific Binding between

Intact IgG-Format Anti-RAS Cytotransmab and GTP-Bound K-RAS

in Cells

FIG. 24 shows the results of confocal microscopy

observation performed to examine whether intact IgG-format

RAS-targeting cytosol-penetrating antibodies would merge with

intracellular H-RAS G12V mutants.

Specifically, fibronectin (Sigma) was coated on a 24

well plate, and then 0.5 ml of a dilution of 2 x 104 NIH3T3

cells expressing mCherry (red fluorescence) H-RAS G12V was

added to each well and incubated at 370C in 5% C02 for 12

hours. Next, the cells were treated with each of 2 pM TMab4,

RT11 and RT11-WYW and incubated at 370C for 12 hours. Next,

the cells were stained in the same manner as described in

Example and were observed with a confocal microscope.

As shown in FIG. 24, with the inner cell membranes in

which red fluorescent activated RAS was located, green

fluorescent RT11 or RT11-WYW was merged, but TMab4 was not

merged.

From the above results, it was found that the intact

IgG-format Ras-targeting cytosol-penetrating antibody did

bind specifically to activated RAS in cells. The degree of

merging was higher in the order of RT11-WYW and RT11.

Example 26: Analysis of Properties of D1-M95 Inducing

Structural Change Depending on pH

For more detailed analysis of the properties of the 1st

amino acid aspartic acid (D) and 9 5 th amino acid methionine

(M) of the light-chain variable region (VL), which induce a

change in the properties of the cytosol-penetrating antibody

depending on pH, mutants were constructed by substituting the

1 't amino acid in the antibody backbone with each of glutamic

acid (E), alanine (A) and asparagine (N) present in the

germline sequences, and substituting the 9 5 th amino acid in

the CDR3 with each of all the 20 amino acids.

When the mutants were constructed, the 87th amino acid

tyrosine was substituted with phenylalanine in order to

increase the protein expression yield that decreased by the

improved endosomal escape motif. Phenylalanine is an amino

acid that can easily interact with the aromatic ring amino

acids and hydrophobic amino acids located in the backbone of

the heavy-chain variable region, thus enhancing the interface

between the light-chain variable region and the heavy-chain

variable region. The light-chain variable region, in which

the 8 7 th amino acid is substituted with phenylalanine and

which has the improved endosomal escape motif WYW at the 9 2 nd,

93 d and 9 4 th amino acid, was named 'hT4-3'. Thus, the cytosol

penetrating intact IgG-format antibody comprising the light

chain variable region was named 'TMab4-3'.

In the same manner as described in Example 1, each of

the mutants was cloned, expressed in HEK293F cell lines, and

purified.

FIG. 25b is a graph showing the results of

quantitatively comparing the number of cells that have taken

up trypan blue depending on pH by mutants constructed by

substituting 95th amino acid methionine of the light-chain

variable region (VL) of a cytosol-penetrating antibody, which

induce a structural change of the cytosol-penetrating

antibody at acidic pH 5.5, with various amino acids.

Specifically, 1 x 104 adherent cells (pgsD-677)

expressing no HSPG receptor were incubated. On the next day,

in the same manner as described in Example 5, the cells were

incubated with each of 1 pM TMab4-3, TMab4-3 D1A, TMab4-3 D1E

and TMab4-3 D1N in 200 pl of each of pH 7.4 buffer

(HBSS(Welgene), 50 mM HEPES pH 7.4)(for maintaining cytosolic

pH) and pH 5.5 buffer (HBSS(Welgene), 50 mM MES pH 5.5)(for

maintaining early endosomal pH) at 370C for 2 hours. After

careful washing with PBS, 200 pl of a mixture of 190 pl of

PBS and 10 pl of trypan blue was added to each well, and the

cells were observed with a microscope. Next, after careful

washing with PBS, the cells were lysed by adding 50 pl of 1%

SDS (sodium dodecyl sulfate) to each well. The cells were

transferred to a 96-well plate, and the absorbance at 590 nm

was measured.

As a result, TMab4-3 DiE showed trypan blue uptake

similar to that of the wild-type, and the TMab4-3 DiA and

TMab4-3 DiN mutants showed reduced trypan blue uptake.

FIG. 25b is a graph showing the results of

quantitatively comparing the number of cells that have taken

up trypan blue depending on pH by mutants constructed by

substituting 95th amino acid methionine of the light-chain

variable region (VL) of a cytosol-penetrating antibody, which

induce a structural change of the cytosol-penetrating

antibody at acidic pH 5.5, with various amino acids.

Specifically, pgsD-677 cells were prepared in the same

manner as described in Example 26. Then, in the same manner

as in described Example 5, the cells were incubated with 1 pM

of each of TMab4-3 and nineteen TMab4-3 mutants in 200 pl of

each of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES pH 7.4

(cytosolic pH)) and pH 5.5 buffer (HBSS(Welgene), 50 mM MES

pH 5.5 (early endosomal pH)) at 370C for 2 hours. After

careful washing with PBS, 200 pl of a mixture of 190 pl of

PBS and 10 pl of trypan blue was added to each well, and the

cells were observed with a microscope. Next, after careful

washing with PBS, the cells were lysed by adding 50 pl of 1%

SDS (sodium dodecyl sulfate) to each well.

The cells were transferred to a 96-well plate, and the

absorbance at 590 nm was measured. As a result, TMab4-3 M95L,

M951 and M95H showed trypan blue uptake similar to that of

TMab4-3, and TMab4-3 M95A, M95S, M95V, M95G and M95P mutants

showed reduced trypan blue uptake. In addition, TMab4-3 M59D

and M59E showed increased pH-dependent trypan blue uptake, but TMab4-3 M95K and M95R mutants showed increased trypan blue uptake at neutral pH.

Therefore, it was found that interaction between

hydrophobic amino acids having long side chains, negatively

charged amino acids, and histidine (H), is most effective for

inducing structural changes at acidic pH.

When the 9 5 th amino acid of the light-chain variable

region is composed of the hydrophobic amino acid methionine

(M), isoleucine (I) or leucine (L), or the negatively charged

amino acid aspartic acid (D) or glutamic acid (E), it is

expected that the carboxylic acid in the side chain of the

negatively charged amino acid will become hydrophobic by

partial protonation, and thus the 9 5 th amino acid will

hydrophobically interacts with aspartic acid (D) or glutamic

acid (E), which is the 1st amino acid of the light-chain

variable region or heavy-chain variable region (Du Z et al.,

2011; Di Russo et al., 2012).

In addition, when the 9 5 th amino acid of the light-chain

variable region is composed of histidine (H), it is expected

that as pH change from 7.4 to 5.5, the net charge of the

amino acid side chains will become positive, and the 95 th

amino acid will induce endosomal escape by electrostatic

interaction with aspartic acid (D) or glutamic acid (E),

which is the 1st amino acid of the light-chain variable region

or heavy-chain variable region.

Example 27: Design of Mutants Introduced with Amino

Acids That 'Induce Change in Properties in Response to pH'

In addition to D1-M95, the present inventors have

attempted to introduce amino acids capable of inducing

endosomal escape by changing their interaction depending on

pH.

Based on the results of structural modeling analysis,

the 9 0 th and 91st amino acids capable of interacting with the

1st amino acid aspartic acid (D) were selected as possible

candidates. To enable interaction under acidic pH conditions,

the 9 0 th amino acid was replaced with histidine (TMab4-3 Q90H),

and the 91st amino acid was substituted with histidine (TMab4

3 Y91H).

In addition, the 91st amino acid capable of additionally

interacting with the 2nd hydrophobic amino acid was

substituted with aspartic acid (TMab4-3 Y91D).

In addition, the 2nd amino acid was also substituted

with negatively charged glutamic acid (E) so that it could

interact with the 1st negatively charged amino acid, and the

9 0 th amino acid was substituted with leucine (L) (TMab4-3 L2E

Q90L) so that it could interact with the 9 5 th hydrophobic

amino acid. Furthermore, the 2 nd amino acid was also

substituted with glutamic acid (E) so that it could interact

with the 1st negatively charged amino acid, and the 97th amino acid was substituted with isoleucine (I) (TMab4-3 L2E T971) so that it could interact with the 9 5 th hydrophobic amino acid.

Table 9 below shows the names and sequences of the

mutants constructed using an overlap PCR technique.

[Table 9]

Name of Variable Sequence SEQ ID NO: Region 1 10 20 abcdef 30 40 50 SEQ ID hT4-3 VL DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYOQKPGKAPKLLIYW 60 70 80 90 100 NO:16 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 ELVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID D1E-M95L VL 60 70 80 90 100 NO:17 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWYWLYTFGOGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID Y91 H VL 60 70 80 90 100 NO:18 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQHWYWMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID Y91DVL 60 70 80 90 100 NO:19 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDWYWMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DEVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID L2E Q90L VL 60 70 80 90 100 NO:20 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQLYWYWMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DEVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID L2E T971 VL 60 70 80 90 100 NO:21 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWYWMYIFGQGTKVEIKR

1 10 20 abcdef 30 40 50 hT4-3 SEQ ID Q90H M95A DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYOQKPGKAPKLLIYW 60 70 80 90 100 NO:22 VL ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYWYWAYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID Q90H VL 60 70 80 90 100 NO:23 ASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHYWYWMYTFGOGTKVEIKR

In the same manner as described in Example 1, each of

the mutants was cloned, expressed in HEK293F cell lines, and

purified.

Example 28: Confirmation of Improvement in Endosomal

Escape Ability of Mutants Introduced with Amino Acids That

'Induce Change in Properties in Response to pH'

FIG. 26a shows a graph showing quantitatively comparing

the number of cells that have taken up trypan blue depending

on pH by mutants designed for the purpose of 'inducing an

additional change in properties in response to pH'.

Specifically, cells were prepared in the same manner as

described in Example 26. Then, in the same manner as

described in Example 5, the cells were incubated with 0.5 or

1 pM of each of seven mutants (including TMab4-3, TMab4-3

D1E-M95L, etc.) in 200 pl of each of pH 7.4 buffer

(HBSS(Welgene), 50 mM HEPES pH 7.4(cytosolic pH)) and pH 5.5

buffer (HBSS(Welgene), 50 mM MES pH 5.5(early endosomal pH))

at 370C for 2 hours. After careful washing with PBS, 200 pl

of a mixture of 190 pl of PBS and 10 pl of trypan blue was

added to each well, and the cells were observed with a

microscope. Next, after careful washing with PBS, the cells

were lysed by adding 50 pl of 1% SDS (sodium dodecyl sulfate)

to each well. The cells were transferred to a 96-well plate,

and the absorbance at 590 nm was measured.

As a result, the TMab4-3 Q90H mutant showed higher

trypan blue uptake than TMab4-3 at 0.5 pM. Additionally,

using the TMab4-3 Q90H mutant showing a significant

difference from the wild-type, an experiment was performed.

FIG. 26b shows a bar graph showing the results of

observing the cytosolic localization of mutants designed for

the purpose of inducing an additional change in properties in

response to pH by confocal microscopy using calcein and

quantifying the calcein fluorescence of the confocal

micrographs.

Specifically, HeLa cells were prepared in the same

manner as described in Example 2, and the cells were

incubated with 0.5 pM and 1 pM of each of TMab4-3 and TMab4-3

Q90H at 370C for 6 hours. After 4 hours, each well containing

PBS or the antibody was treated with 150 pM calcein and

incubated at 370C for 2 hours. The cells were washed with PBS

and weakly acidic solution in the same manner as described in

Example 2, and then fixed. The nucleus was blue-stained with

Hoechst33342 and observed with a confocal microscope.

As a result, in the cells treated with TMab4-3 Q90H,

green calcein fluorescence localized in the cytosol increased

compared to that in the cells treated with TMab4-3. Therefore,

it was confirmed that, in addition to the 9 5 th amino acid of

the light-chain variable region of the cytosol-penetrating

antibody, the 9 0 th amino acid interacted with the 1 't amino acid and induced endosomal escape by a pH-dependent change in the interaction.

Table 10 below the CDR3 sequence of the light-chain

variable region of the mutant having an increased ability to

escape from endosomes by inducing an additional change in the

properties depending on pH.

[Table 10]

Name of

Light SEQID Chain CDR3 Sequence NO: Variable Region

Kabat No.

hT4 3 SEQ ID Q90H VL Q H Y W Y WM Y T _____ ____ ____ NO:24

Example 29: Investigation of Endosomal Escape Ability at

Varying Lengths of CDR3 of Light-Chain Variable Region

85% or more of the CDR3 of the light-chain variable

region consists of 9 amino acids. Depending on the number and

composition of amino acids, the CDR3 loop structure varies.

In the present disclosure, to analyze how the endosomal

escape ability changes depending on the number and composition of amino acids, mutants comprising a CDR3 consisting of 8, 10 or 11 amino acids were constructed.

Table 11 below shows the names and sequences of the

mutants constructed using an overlap PCR technique.

[Table 11]

Nameof Variable Sequence SEQ ID NO: Region 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYOOKPGKAPKLLIYW SEQ lID L8-1 VL 60 70 80 90 100 NO:25 ASTRFSGVPSRFSGSGSGTDFTLTISSLPEDFATYFCQQYWYWMTFGDGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYOOKPGKAPKLLIYW SEQ ID L8-2 VL 60 70 80 90 100 NO:26 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYFCQQWYWMPTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID L10-1 VL 60 70 80 90 100 NO:27 ASTRESGVPSRFSGSGSGTDFTLTISSLPEDFATYFCQQYWYWPMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYOOKPGKAPKLLIYW SEQ ID L10-2 VL 60 70 80 90 100 NO:28 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQYWYWLMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYQOKPOKAPKLLIYW SEQ lID L10-3 VL 60 70 80 90 100 NO:29 ASTRESGVPSRFSGSGSGTDFTLTISSLPEDFATYYCQQYWYWYMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQOKPGKAPKLLIYW SEQ ID 111-1 VL 60 70 80 90 100 NO:30 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQYWYWLYMYTF3QGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID 11-2 VL 60 70 80 90 100 NO:31 ASTRESGVPSRFSGSGSGTDFTLTISSLPEDFATYYCQQYPWYWPMYTFGQGTKVEIKR

In the same manner as described in Example 1, each of

the mutants was cloned, expressed in HEK293F cell lines, and

purified.

FIG. 27 is a graph quantitatively comparing the number

of cells that taken up trypan blue depending on pH by mutants

obtained by changing the amino acid number of the CDR3 of the

light-chain variable region of a cytosol-penetrating antibody.

Specifically, cells were prepared in the same manner as

described in Example 26. Then, in the same manner as

described in Example 5, the cells were incubated with 1 pM of

each of seven mutants (including TMab4-3, TMab4-3 L8-1, etc.)

in 200 pl of each of pH 7.4 buffer (HBSS(Welgene), 50 mM

HEPES pH 7.4(cytosolic pH)) and pH 5.5 buffer (HBSS(Welgene),

50 mM MES pH 5.5(early endosomal pH)) at 370C for 2 hours.

After careful washing with PBS, 200 pl of a mixture of 190 pl

of PBS and 10 pl of trypan blue was added to each well, and

the cells were observed with a microscope. Next, after

careful washing with PBS, the cells were lysed by adding 50

pl of 1% SDS (sodium dodecyl sulfate) to each well. The cells

were transferred to a 96-well plate, and the absorbance at

590 nm was measured.

As a result, in comparison with TMab4-3, the mutants

showed increased trypan blue uptake at neutral pH as the

number of amino acids increased. The reason is believed to

be as follows. As the number of amino acids increases, the overall CDR3 loop structure is stretched, and the endosomal escape motif WYW which binds to the cell membrane in order to escape from endosomes is exposed to the outside, and thus trypan blue uptake increases even at neutral pH.

In addition, in the experimental results, it was

confirmed that even when the distance between the 9 5 th amino

acid, which induces endosomal escape by a pH-dependent change

in interaction, and the 9 2 nd, 9 3 rd or 9 4 th amino acid, which

influences endosomal escape by binding to the phospholipid,

increases, the properties of the endosomal escape motif are

maintained.

Example 30: Logic of Possibility of Imparting Improved

Endosomal Escape Motif to Light-Chain Variable Region of

Conventional Therapeutic Antibody

Currently commercially available therapeutic antibodies

include many kinds of monoclonal antibodies that target cell

surface receptors, particularly cell surface receptors that

undergo endocytosis. However, these conventional antibodies

have disadvantages in that their binding to antigen is not

broken after endocytosis, and these antibodies do not

localize in the cytosol and are released out of the cells

because they have no endosomal escape ability. Thus, if

endosomal escape ability can be imparted to these receptor

targeting antibodies that undergo endocytosis, there is an advantage in that these antibodies can be used in a wider range of applications.

In addition, the use of the stable backbone of

commercially available therapeutic antibodies can increase

the overall expression yield, and when the affinity of these

antibodies for HSPG, a non-tumor-specific receptor, is

eliminated, tumor tissue specificity can be imparted to these

antibodies.

To impart an improved endosomal escape motif, the

sequences of the light-chain variable regions of receptor

targeting antibodies that undergo endocytosis were compared

with the sequence of the light-chain variable region of the

cytosol-penetrating antibody. As a result, candidate light

chain variable regions were selected, which have a negatively

charged amino acid as the 1 't amino acid and in which

backbone amino acids that can influence the CDR3 loop

structure are the same as those of the cytosol-penetrating

antibody.

Mutants were constructed by the CDR3 sequences of the

candidate light-chain variable regions with the CDR3 sequence

of the cytosol-penetrating antibody.

Table 12 shows the names and sequences of the mutants

constructed using a genesis synthesis technique.

[Table 12]

Name of Sequence SEQ ID NO: Variable Region 1 10 20 abcd 40 50 Necitumumab- EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD SEQ ID WYW VL 60 70 80 90 100 NO:32 ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQYWYWMYTFGQGTKAEIKR 1 10 20 abcd 40 50 Panitumumab- DIQMTOSPSSLSASVGDRVTITCOASQDISNYLNWYQQKPGKAPKLLIYD SEQ ID WYW VL 60 70 80 90 100 NO:33 ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQYWYWMYTFGGGTKVEIKR 1 10 20 abcdef 30 40 50 Lumretuzumab- DIVMTQSPDSLAVSLGERATINCKSSQSVLNSGNQKNYLTWYQQKPGQAPKLLIYW SEQ ID WYW VL 60 70 80 90 100 NO:34 ASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQYWYWMYTFGQGTKLEIKR 1 10 20 abcdef 30 40 50 Nimotuzumab- DIQMTQSPSSLSASVGDRVT ITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYK SEQ ID WYW VL 60 70 80 90 100 N0:35 VSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQQYWYWMYTFGQGTKLQITR 1 10 20 abcd 40 50 Emibetuzumab- DIQMTOSPSSLSASVGDRVTITCSVSSSVSSIYLHWYQQKPGKAPKLLIYS SEQ ID WYW VL 60 70 80 90 100 NO:36 TSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGGGTKVEIKR 1 10 20 abcd 40 50 Pertuzumab- DIQMTOSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYS SEQ ID WYW VL 60 70 80 90 100 NO:37 ASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYWYWMYTFGQGTKVEIKR

FIG. 28a shows a process of constructing an intact IgG

format RAS-targeting cytosol-penetrating antibody in which an

improved endosomal escape motif is introduced into the light

chain variable region of a conventional therapeutic antibody.

As shown in FIG. 28a, in the same manner as described in

Example 1, cloning of the light-chain variable region was

performed, and the resulting animal expression vector and he

animal expression vector encoding the heavy chain comprising

the heavy-chain variable region that binds specifically to

GTP-bound K-RAS were transiently co-transfected into HEK293F

protein-expressing cells. Next, purification of the resulting

intact IgG-format anti-RAS cytotransmab was performed in the

same manner as described in Example 1.

Example 31: Confirmation of Possibility of Imparting

Improved Endosomal Escape Motif to Light-Chain Variable

Region of Conventional Therapeutic Antibody

FIG. 28b shows the results of fluorescence microscopic

observation performed to examine whether the HSPG binding

affinity and cytosol-penetrating ability of an intact IgG

format RAS-targeting cytosol-penetrating antibody in which an

improved endosomal escape motif is introduced into the light

chain variable region of a conventional therapeutic antibody

would be reduced or eliminated.

Specifically, HeLa cells were prepared in the same

manner as described in Example 2. When the cells were

stabilized, the cells were incubated with PBS or each of 1 pM

RT11-3, RT11-Neci-WYW, RT11-Nimo-WYW, RT11-Pani-WYW, RT11

Pert-WYW, RT11-Lumr-WYW and RT11-Emib-WYW at 370C for 6 hours.

The cells were washed with PBS and weakly acidic

solution in the same manner as described in Example 2, and

then subjected to cell fixation, cell perforation and

blocking processes. Each of the antibodies was stained with

an FITC (green fluorescence)-labeled antibody that specifically recognizes human Fc. The nucleus was blue stained with Hoechst33342 and observed with a confocal microscope. As a result, in all the six intact IgG-format

RAS-targeting cytosol-penetrating antibodies comprising the

monoclonal antibody backbone imparted with the improved

endosomal escape motif, no fluorescence was observed.

FIG. 28c shows a graph quantitatively comparing the

number of cells that taken up trypan blue at acidic pH by an

intact IgG-format RAS-targeting cytosol-penetrating antibody

in which an improved endosomal escape motif is introduced

into the light-chain variable region of a conventional

therapeutic antibody.

Specifically, Ramos cells were attached to plates in the

same manner as described in Example 5. Then, the cells were

incubated with each of 1 pM RT11-3, RT11-Neci-WYW, RT11-Nimo

WYW, RT11-Pani-WYW, RT11-Pert-WYW, RT11-Lumr-WYW, and RT11

Emib-WYW in 200 pl of pH 7.4 buffer (HBSS(Welgene), 50 mM

HEPES pH 7.4(cytosolic pH)) and pH 5.5 buffer(HBSS(Welgene),

50 mM MES pH 5.5(early endosomal pH)) at 370C for 2 hours.

After careful washing with PBS, 200 pl of a mixture of 190 pl

of PBS and 10 pl of trypan blue was added to each well, and

the cells were observed with a microscope.

The number of cells showing trypan blue uptake was

counted and expressed as percentage relative to the total

number of cells. A total of 400 or more cells were counted, and the mean values are graphically shown. As a result, except for RT11-Pert, the five intact IgG-format RAS targeting cytosol-penetrating antibodies comprising the therapeutic antibody backbone imparted with the improved endosomal escape motif showed trypan blue uptake similar to that of RT11-3.

Example 32: Confirmation of Maintenance of Specific

Binding between Intact IgG-Format RAS-Targeting Cytosol

Penetrating Antibody Comprising Therapeutic Antibody Backbone

Imparted with Improved Endosomal Escape Motif and GTP-Bound

K-RAS

FIG. 29a shows the results of ELISA performed to measure

the affinities of an intact IgG-format RAS-targeting cytosol

penetrating antibody, in which an improved endosomal escape

motif is introduced into the light-chain variable region of a

conventional therapeutic antibody, for GppNHp-bound K-RAS

G12D and GDP-bound K-RAS G12D, which are K-RAS mutants.

Specifically, each of a GppNHp-bound K-RAS G12D and a

GDP-bound K-RAS G12D, which are target molecules, was

incubated in 96-well EIA/RIA plates (COSTAR Corning) at 37°C

for 1 hour, followed by washing three times with 0.1% PBST

(0.1 % Tween20, pH 7.4, 137 mM NaCl, 12mM phosphate, 2.7 mM

KCl) (SIGMA) for 10 minutes. Each well was incubated with 5%

PBSS (5% Skim milk, pH7.4, 137 mM NaCl, 12mM phosphate, 2.7 mM KCl) (SIGMA) for 1 hour, and then washed three times with

0.1% PBST for 10 minutes. Next, each well was incubated with

each of the IgG-format RAS-targeting cytosol-penetrating

antibodies (RT11-3, RT11-Neci-WYW, RT11-Nimo-WYW, RT11-Pani

WYW, RT11-Pert-WYW, RT11-Lumr-WYW, RT11-Emib-WYW), and then

washed three times with 0.1% PBST for 10 minutes. As a marker

antibody, goat alkaline phosphatase-conjugated anti-human mAb

(SIGMA) was used. Each well was treated with pNPP (p

nitrophenyl palmitate) (Sigma), and the absorbance at 405 nm

was measured.

Affinities for the K-RAS mutants were analyzed. As a

result, except for RT11-Nimo, the five intact IgG-format RAS

targeting cytosol-penetrating antibodies comprising the

therapeutic antibody backbone imparted with the endosomal

escape motif showed no difference in affinity from RT11-3,

and all the clones did not bind to the GDP-bound K-RASs used

as negative controls.

FIG. 29b shows a schematic view showing a process of

constructing an intact IgG-format RAS-targeting cytosol

penetrating antibody in which an improved endosomal escape

motif is introduced into the RGD10 peptide-fused light-chain

variable region of a conventional therapeutic antibody.

Because the intact IgG-format RAS-targeting cytosol

penetrating antibody imparted with the improved endosomal

escape motif showed no cell-penetrating ability, an RGD10 peptide specific for integrin avB3 which is overexpressed in neovascular cells and various tumors was genetically fused to the N-terminus of the light chain by two GGGGS linkers. The

RGD10 peptide has an affinity similar to that of a RGD4C

peptide, but has characteristics in that it has a single

disulfide bond formed by two cysteine residues and can be

genetically fused.

In addition, based on the results of analysis of

expression yield, endosomal escape ability, and affinity for

Ras, the RGD10 peptide was fused to the N-terminus of the

light-chain variable region of each of RT11-Pani-WYW and

RT11-Neci-WYW which are excellent candidate antibodies.

FIG. 29c shows the results of confocal microscopy

performed to examine whether an intact IgG-format RAS

targeting cytosol-penetrating antibody in which an improved

endosomal escape motif is introduced into the RGD10 peptide

fused light-chain variable region of a conventional

therapeutic antibody would merge with intracellular activated

H-RAS G12V mutants.

Specifically, 0.5 ml of a dilution of 2 x 104 human

colorectal cancer SW480 cells having a K-RAS G12V mutation

were added to each of a 24-well plate and incubated with each

of 1 pM RT11-i3, RT11-i-Neci-WYW and RT11-i-Pani-WYW at 370C

in 5% C02 for 12 hours. Next, in the same manner as described

in Example 2, antibody labeling and nucleus staining were performed, and the cells were treated with a Ras-labeled antibody at 370C for 1 hour. Then, the cells were secondary antibody and observed with a confocal microscope.

With the inner cell membrane in which the red

fluorescent activated RAS was located, green fluorescent

RT11-i3, RT11-i-Neci-WYW and RT11-i-Pani-WYW were merged.

The above experimental results indicate that the intact

IgG-format RAS-targeting cytosol-penetrating antibody

introduced with the improved endosomal escape motif binds

specifically to activated RAS in cells.

Example 33: Logic of Possibility of Imparting Improved

Endosomal Escape Motif to CDR of Heavy-Chain Variable Region

The heavy-chain variable region and the light-chain

variable region are structurally common in that they have a

beta-sheet structure as a backbone and are composed of three

CDR having a loop structure. Thus, it was considered that the

endosomal escape motif of the light-variable variable region,

which induces endosomal escape by a pH-dependent change in

interaction, can also be applied to the heavy-chain variable

region.

Whether this phenomenon is reproducible in the heavy

chain variable region was analyzed through the sequence and

three-dimensional structure of the heavy-chain variable

region. As a result, the endosomal escape motif could be grafted into the CDR3 at a distance that can interact with the 1 't amino acid glutamic acid (E) of the heavy-chain variable region.

The number of amino acids in the CDR3 of the wild-type

heavy-chain variable region is 11, and the center of the loop

structure of the CDR3 is significantly exposed to the surface.

For this reason, it is considered that the pH-dependent

phenomenon occurring in the light-chain variable region

hardly occurs. For this reason, the amino acid number of the

CDR3 was reduced to 7 or 8 while maintaining a portion of the

sequence.

In addition, it was considered that an amino acid

capable of interacting with the 1 't amino acid of the heavy

chain variable region at early endosomal pH is the 1 0 2 nd amino

acid of the heavy-chain variable region and this amino acid

is located at a suitable distance. Thus, this amino acid was

substituted with leucine (L).

Mutants were constructed by introducing the improved

endosomal escape motif into the CDR3 of the heavy-chain

variable region.

Tables 13, 14 and 15 show the heavy-chain variable

region sequences obtained by grafting the designed endosomal

escape motif into the heavy-chain variable region. Table 13

below shows the full-length sequences of the human antibody

light-chain variable regions according to the Kabat numbering system, and Tables 14 and 15 below show the CDR1 and CDR2 sequences or CDR3 sequences of the antibody sequences shown in Table 13.

[Table 13]

Name of Variable Sequence SEQ ID NO: Region 1 10 20 30 40 50 SEQ ID HTO VH EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYVMHWVRAPGKGLEWVSAINPYNDGNYY 60 70 80 90 110 N0:38 ADSVKGRFTISRDNSRKTLYLMNSLRAEDTAVYYCARGAYKRGYAMDYWGOGTTVTVSS 1 10 20 30 40 50 EVQLVESGGGLVQPGGSLRLSCAASYTFTSYVMHWVRQAPGKGLEWVSAINPYNDGNYY SEQ ID HTO-01 VH 60 70 80 90 110 NO:39 ADSVKGRFTISRDNSRKTLYLMNSLRAEDTAVYYCARGWYWMDLWGDGTTVTVSS 1 10 20 30 40 50 EVQLVESGGGLV0PGGSLRLSCAASGYTFTSYVMHWVROAPGKGLEWVSAINPYNDGNYY SEQ ID HTO-02 VH 60 70 80 90 110 N0:40 ADSVKGRFTISRDNSRKTLYLOMNSLRAEDTAVYYCARGWYWFDLWGQGTTVTVSS 1 10 20 30 40 50 SEQ ID HTO-03 VH EVQLVESGGGLVOPGGSLRLSCAASGYTFTSYVMHWVRQAPGKGLEWVSAINPYNDGNYY 60 70 80 90 110 N0:41 ADSVKGRFTISRDNSRKTLYLQMNSLRAEDTAVYYCARGWYWGFDLWGGTTVTVSS 1 10 20 30 40 50 SEQ ID HTO-04 VH EVQLVESGGGLVOPGGSLRLSCAASGYTFTSYVMHWVRQAPGKGLEWVSAINPYNDGNYY 60 70 80 90 110 N0:42 ADSVKGRFTISRDNSRKTLYLQMNSLRAEDTAVYYCARYWYWMDLWGQGTTVTVSS

[Table 14]

CDR1 CDR2 Sequence ___ ___ __ Sequence _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

Kabat No. 4-n" rn'Mt2m o L-n 14) "I d)n n LOnl 10 I) 1) w W tD CO

Heavy Chain Variable S Y V M H A I N P Y N D G N Y Y A D S V K G Region

SEQ ID NO: SEQID SEQ ID NO:44 I NO:43

[Table 15]

Name of Heavy SEQ ID Chain CDR3 Sequence NO:

Variable Region

Kabat No. mm O)O O2 o08.

G A R K R G Y A M D Y SEQ ID 10HTO VH NO.45 SEQ ID HTO-01 VH G W Y W M - - - - D L NO:46 SEQ ID HTO-02 VH G W Y W F - - - - D L NO:47 SEQ ID HTO-03 VH GWYWG F - - - D L NO:48 SEQ ID HTO-04VH Y W Y W M - - - - D L I NO:49

FIG. 30a shows a process of constructing a cytosol

penetrating antibody having a light-chain variable region from which endosomal escape ability is removed and a heavy chain variable region into which an improved endosomal escape motif is introduced.

In order to evaluate the endosomal escape ability of the

heavy-chain variable region introduced with the improved

endosomal escape motif, the 9 2nd to 9 4 th amino acids (WYW) of

the light-chain variable region, which are involved in

endosomal escape, AAA (three consecutive alanines), thereby

removing the function thereof. In the same manner as

described in Example 1, cloning of the heavy-chain variable

region was performed, and the resulting heavy chain together

with the light chain comprising the light-chain variable

region from which the endosomal escape motif has been removed

was expressed in HEK293F cell lines and purified.

In order to more clearly name the cytosol-penetrating

antibody comprising the heavy-chain variable region

introduced with the improved endosomal escape motif, TMab4 is

abbreviated as CT. In other words, TMab4-AAA is CT-AAA.

Example 34: Confirmation of Possibility of Imparting

Improved Endosomal Escape Motif to CDR of Heavy-Chain

Variable Region

FIG. 30b shows a graph quantitatively comparing the

number of cells that have taken up trypan blue depending on

pH by a cytosol-penetrating antibody having a light-chain variable region from which endosomal escape ability is removed and a heavy-chain variable region into which an improved endosomal escape motif is introduced.

Specifically, as shown in FIG. 30b, Ramos cells were

attached to plates in the same manner as described in Example

5. Then, the cells were incubated with each of 1 pM TMab4-WYW,

TMab4-AAA, CT01-AAA, CT02-AAA, CT03-AAA and CT04-AAA in 200

pl of each of pH 7.4 buffer(HBSS(Welgene), 50 mM HEPES pH

7.4(cytosolic pH)) and pH 5.5 buffer (HBSS(Welgene), 50 mM

MES pH 5.5(early endosomal pH)) at 370C for 2 hours. After

careful washing with PBS, 200 pl of a mixture of 190 pl of

PBS and 10 pl of trypan blue was added to each well, and the

cells were observed with a microscope. The number of cells

showing trypan blue uptake was counted and expressed as

percentage relative to the total number of cells. A total of

400 or more cells were counted, and the mean values are

graphically shown. It was observed that the CT01-AAA, CT02

AAA, CT03-AAA and CTO4-AAA all showed trypan blue uptake

equal to about half of TMab4-3. However, it was shown that

the CTO4-AAA mutant showed trypan blue uptake even at neutral

pH.

FIG. 30c shows the results of confocal microscopy

performed to observe the GFP fluorescence by enhanced split

GFP complementation of a GFP11-SBP2-fused cytosol-penetrating

antibody having a light-chain variable region from which endosomal escape ability has been removed, and a heavy-chain variable region into which an improved endosomal escape motif has been introduced.

Specifically, transformed HeLa cells stably expressing

SA-GFP1-10 were prepared in the same manner as described in

Example 2. When the cells were stabilized, the cells were

incubated with PBS or each of 1.6 pM CT01-AAA-GFP11-SBP2,

CT02-AAA-GFP11-SBP2, CT03-AAA-GFP11-SBP2 and CT04-AAA-GFP11

SBP2 at 370C for 6 hours. The cells were washed with PBS and

weakly acidic solution in the same manner as described in

Example 2, and then fixed. The nucleus was blue-stained with

Hoechst33342 and observed with a confocal microscope. As a

result, green fluorescence was observed in the cells with

CT01-AAA-GFP11-SBP2, CT02-AAA-GFP11-SBP2, CT03-AAA-GFP11-SBP2

or CT04-AAA-GFP11-SBP2.

FIG. 30d shows the results of confocal microscopy

performed using calcein in order to observe the cytosolic

localization of a cytosol-penetrating antibody having a

light-chain variable region from which endosomal escape

ability has been removed and a heavy-chain variable region

into which an improved endosomal escape motif has been

introduced.

Specifically, HeLa cells were prepared in the same

manner as described in Example 2. The cells were incubated

with each of 0.2 pM and 1 pM CT01-AAA, CT02-AAA and CT03-AAA at 370C for 6 hours. After 4 hours, each well containing PBS or the antibody was treated with 150 pM calcein and incubated at 370C for 2 hours. In the same manner as descried in

Example 2, the cells were washed with PBS and weakly acidic

solution, and then fixed. The nucleus was blue-stained with

Hoechst33342 and observed with a confocal microscope. In the

cells treated with CT01-AAA or CT02-AAA, green calcein

fluorescence localized in the cytosol was similar to that in

the cells treated with TMab4. However, in the cells treated

with CT03-AAA, green calcein fluorescence localized in the

cytosol was weaker than that in the cells treated with CT.

Therefore, it was confirmed that even when the improved

endosomal escape motif is imparted to the heavy-chain

variable region, the antibody can escape from endosomes and

finally localize in the cytosol.

Example 35: Analysis of Properties of E1-L102 of Heavy

Chain Variable Region That Induces Structural Change

Depending on pH

For more detailed analysis of the 1st amino acid

glutamic acid and 102nd amino acid leucine of the heavy-chain

variable region, which induce a structural change depending

on pH, mutants were constructed by substituting the 1st amino

acid in the antibody backbone with each of aspartic acid,

alanine and glutamine present in germline sequences, and substituting the 1 0 2nd amino acid in the CDR3 with each of 12 amino acids of the light-chain variable region, which showed trypan blue uptake at neutral or acidic pH. In the same manner as described in Example, each of the mutants was cloned, expressed in HEK293F cell lines, and purified.

FIG. 31a is a graph quantitatively comparing the number

of cells that taken up trypan blue depending on pH by mutants

constructed by substituting the 1 't amino acid glutamic acid

of the heavy-chain variable region (VH) of a cytosol

penetrating antibody, which is involved in induction of a

structural change in properties of the antibody at acidic pH

5.5, with various amino acids.

Specifically, 1x10 4 pgsD-677 cells were incubated in 24

well plates in the same manner as described in Example 26. On

the next day, in the same manner as described in Example 5,

the cells were incubated with each of 1 pM CT01-AAA, CT01-AAA

ElA, CT01-AAA ElD and CT01-AAA E1Q in 200 pl of each of pH

7.4 buffer (HBSS(Welgene), 50 mM HEPES pH 7.4(cytosolic pH))

and pH 5.5 buffer(HBSS(Welgene), 50 mM MES pH 5.5(early

endosomal pH)) at 37°C for 2 hours. After careful washing

with PBS, 200 pl of a mixture of 190 pl of PBS and 10 pl of

trypan blue was added to each well, and the cells were

observed with a microscope. Next, after careful washing with

PBS, the cells were lysed by adding 50 pl of 1% SDS (sodium

dodecyl sulfate) to each well. The cells were transferred to a 96-well plate, and the absorbance at 590 nm was measured.

As a result, CT01-AAA ElD showed trypan blue uptake similar

to that of the wild type, and the CT01-AAA ElA and CT01-AAA

E1Q mutants showed reduced trypan blue uptake compared to

that of the wild type.

FIG. 31a is a graph quantitatively comparing the number

of cells that taken up trypan blue depending on pH by mutants

constructed by substituting 1 0 2 "d amino acid leucine of the

heavy-chain variable region (VH) of a cytosol-penetrating

antibody, which is involved in induction of a structural

change of the antibody at acidic pH 5.5, with various amino

acids.

Specifically, pgsD-677 cells were prepared in the same

manner as described in Example 26. Then, in the same manner

as in described Example 5, the cells were incubated with 1 pM

of each of CT01-AAA and nineteen CT01-AAA L102X mutants in

200 pl of each of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES

pH 7.4 (cytosolic pH)) and pH 5.5 buffer (HBSS(Welgene), 50

mM MES pH 5.5 (early endosomal pH)) at 370C for 2 hours.

After careful washing with PBS, 200 pl of a mixture of 190 pl

of PBS and 10 pl of trypan blue was added to each well, and

the cells were observed with a microscope. Next, after

careful washing with PBS, the cells were lysed by adding 50

pl of 1% SDS (sodium dodecyl sulfate) to each well. The cells

were transferred to a 96-well plate, and the absorbance at

590 nm was measured. As a result, compared to CT01-AAA, CT01

AAA L1021, L102M, and L102H showed trypan blue uptake similar

to that of the wild type, and the CT01-AAA L102K and L102R

mutants showed increased trypan blue uptake at neutral pH.

This suggests that interaction between hydrophobic amino

acids having long side chains, negatively charged amino acids,

and histidine (H), is most effective so that the 1 0 2 nd amino

acid of the heavy-chain variable region induces endosomal can

escape through a change in interaction at early endosomal pH

5.5, like the 9 5 th amino acid of the light-chain variable

region.

Example 36: Construction of Endosomal Escape Motif

Mutants Having Three Tryptophan Residues

In order to improve the endosomal escape ability of the

endosomal escape motif having two tryptophan residues, an

endosomal escape motif having a total of three tryptophan

residues was constructed by substituting the 9 2nd to 9 4 th amino

acids with tryptophan.

Tables 16 and 17 show light-chain variable region mutant

sequences obtained by introducing the endosomal escape motif

having three tryptophan residues. Specifically, Table 16

below shows the full-length sequence of the human antibody

light-chain variable region according to the Kabat numbering system, and Table 17 below the CDR3 sequence of the antibody sequence shown in Table 16.

[Table 16]

Name of Variable Sequence SEQ ID NO: Region 1 10 20 abcdef 30 40 50 SEQ ID hT4-3VL DLVMTOSPSSLSASVGDRVTITCKSSOSLFNSRTRKNYLAWYQQKPGKAPKLLIYW 60 70 80 90 100 No:16 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYFCQOYWYWMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 hT4-3 DLVMTQSPSSLSASVGDRVTITCKSSQSLFNSRTRKNYLAWYQQKPGKAPKLLIYW SEQ ID WWW VL 60 70 80 90 100 N0:50 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYFCQQYVMYTFGQGTKVEIKR

[Table 17]

Name of

Light SEQID Chain CDR3 Sequence NO: Variable Region

Kabat No. hT43 SEQ ID WWWVL QQYWWWMYT _____ ____ ____ NO:51

Tables 18 and 19 show heavy-chain variable region mutant

sequences obtained by introducing the endosomal escape motif

having three tryptophan residues. Specifically, Table 18

below shows the full-length sequence of the human antibody light-chain variable region according to the Kabat numbering system, and Table 19 below the CDR3 sequence of the antibody sequence shown in Table 18.

[Table 18]

Name of Variable Sequence SEQ ID NO: Region 1 10 20 30 40 50 EVQLVESGGGLVOPGGSLRLSCAASGYTFTSYVMHWVRQAPGKGLEWVSAINPYNDGNYY SEQ ID HTO-01 VH 60 70 80 90 110 N0:38 ADSVKGRFTISRDNSRKTLYLOMNSLRAEDTAVYYCARGWYWMDLWGDGTTVTVSS 1 10 20 30 40 50 HTO-01 EVOLVESGGGLVOPGGSLRLSCAASGYTFTSYVMHWVRQAPGKGLEWVSAINPYNDGNYY SEQ ID WWW VH 60 70 80 90 110 N0:52 ADSVKGRFTISRDNSRKTLYLQMNSLRAEDTAVYYCARGWMDLWGQGTTVTVSS

[Table 19]

Name of Heavy SEQ ID Chain CDR3 Sequence NO:

Variable Region

Kabat No. c

HTO-01 SEQ ID WWW VH GWWWM- - - - DL NO:53

In order to evaluate the endosomal escape ability of the

heavy-chain variable region or heavy-chain variable region

comprising the endosomal escape motif having three tryptophan

residues, this heavy-chain variable region or heavy-chain variable region and the heavy-chain variable region or light variable region that does not comprise the endosomal escape motif were expressed together in HEK293F cell lines in the same manner as described in Example 1, and purified.

Example 37: Confirmation of Improvement in Endosomal

Escape Ability of Intact IgG-Format Cytosol-Penetrating

Antibody Comprising the Heavy-Chain Variable Region or Light

Chain Variable Region Introduced with Endosomal Escape Motif

Having Two or Three Tryptophan Residues

As one strategy for improving the endosomal escape

ability, the endosomal escape motif was imparted to both the

heavy-chain variable region and the light-chain variable

region. Thus, a single intact IgG-format cytosol-penetrating

antibody includes a total of four endosomal escape motifs.

The heavy-chain variable region and the light-chain

variable region, which comprise the endosomal escape motif

having two or three tryptophan motifs, were expressed

together in HEK293F cell lines in the same manner as

described 1 above, and purified.

FIG. 32a shows a graph quantitatively comparing the

number of cells that have taken up trypan blue depending on

pH by intact IgG-format cytosol-penetrating antibodies having

a light-chain variable region and/or a heavy-chain variable region introduced with an endosomal escape motif having three tryptophan residues.

Specifically, pgsD-677 cells were prepared in the same

manner as described in Example 26. Then, in the same manner

as in described Example 5, the cells were incubated with 0.5

or 1 pM of each of CT-3, CT-3_WWW, CT01-AAA, CT01_WWW-AAA,

and CT01-3, and CT01_WWW-3_WWW in 200 pl of each of pH 7.4

buffer (HBSS(Welgene), 50 mM HEPES pH 7.4 (cytosolic pH)) and

pH 5.5 buffer (HBSS(Welgene), 50 mM MES pH 5.5 (early

endosomal pH)) at 370C for 2 hours. After careful washing

with PBS, 200 pl of a mixture of 190 pl of PBS and 10 pl of

trypan blue was added to each well, and the cells were

observed with a microscope. Next, after careful washing with

PBS, the cells were lysed by adding 50 pl of 1% SDS (sodium

dodecyl sulfate) to each well. The cells were transferred to

a 96-well plate, and the absorbance at 590 nm was measured.

As a result, CT-3_WWW, CT01_WWW-AAA and CT01_WWW-3_WWW

showed significantly increased trypan blue uptake, compared

to the CT-3, CT01-AAA and CT01-3 comprising the existing

endosomal escape motif. In addition, compared to CT-3 and

CT01-AAA, the CT01-3 and CT01_WWW-3_WWW comprising the

endosomal escape motif in both the heavy-chain and light

chain variable regions showed higher trypan blue uptake.

FIG. 32b shows a bar graph showing the results of

observing the cytosolic localization of intact IgG-format cytosol-penetrating antibodies having a light-chain variable region and/or a heavy-chain variable region introduced with an endosomal escape motif having three tryptophan residues by confocal microscopy using calcein and quantifying the calcein fluorescence of the confocal micrographs.

Specifically, HeLa cells were prepared in the same

manner as described in Example 2. The cells were incubated

with 0.25, 0.5 and 1 pM of each of CT-3, CT-3_WWW, CT01-AAA,

CT01_WWW-AAA, CT01-3, and CT01_WWW-3_WWW at 370C for 6 hours.

After 4 hours, each well containing PBS or the antibody was

treated with 150 pM calcein and incubated at 370C for 2 hours.

In the same manner as described in Example 2The cells were

washed with PBS and weakly acidic solution and fixed. The

nucleus was blue-stained with Hoechst33342 and observed with

a confocal microscope.

As a result, compared to the cells treated with the CT-3,

CT01-AAA or CT01-3 comprising the existing endosomal escape

motif, the cells treated with CT-3_WWW, CT01_WWW-AAA or

CT01_WWW-3_WWW showed stronger green calcein fluorescence

that localized in the cytosol. In addition, compared to the

cells treated with CT-3 or CT01-AAA, the cells treated with

the CT01-3 or CT01_WWW-3_WWW comprising the endosomal escape

motif in both the heavy-chain and light-chain variable

regions showed stronger green calcein fluorescence that

localized in the cytosol.

It was confirmed that the endosomal escape motif having

three tryptophan motifs has improved endosomal escape motif

compared to the existing endosomal escape motif, and even

when the endosomal escape motif was imparted to the heavy

chain variable region and the light-chain variable region,

the endosomal escape ability was improved.

Example 38: Confirmation of Improvement in Endosomal

Escape Ability of Intact IgG-Format Cytosol-Penetrating

Antibody Comprising Heavy-Chain Variable Region Introduced

with Improved Endosomal Escape Motif and Light-Chain Variable

Region Having Therapeutic Antibody Backbone Imparted with

Improved Endosomal Escape Ability

In order to confirm that the endosomal escape ability is

improved when the endosomal escape motif is imparted to both

the heavy-chain variable region and the light-chain variable

region, the heavy-chain variable region introduced with the

improved endosomal escape motif and the light-chain variable

region having the therapeutic antibody backbone imparted with

endosomal escape ability were expressed together in HEK293F

cell lines and purified.

Specifically, the intact IgG-format cytosol-penetrating

antibody comprising the heavy-chain variable region

introduced with the improved endosomal escape motif and the

light-chain variable region imparted with improved endosomal ability showed no cell penetrating ability. For this reason, an EpCAM-targeting cyclic peptide specific for EpCAM which is overexpressed on the cell membrane surface in various tumors including colorectal cancer was genetically fused to the N terminus of the antibody by two GGGGS linkers so that the antibody could penetrate cells (US 2015/0246945 Al).

FIG. 33a shows a schematic view showing a process of

constructing an intact IgG-format cytosol-penetrating

antibody in which an improved endosomal escape motif has been

introduced into a heavy-chain variable region thereof and an

improved endosomal escape motif has been introduced into a

light-chain variable region of a conventional therapeutic

antibody fused with an EpCAM-targeting peptide.

Specifically, animal expression vectors encoding a heavy

chain comprising the heavy-chain variable region introduced

with the improved endosomal escape motif and a light chain

comprising the monoclonal antibody light-chain variable

region imparted with improved endosomal ability were

transiently co-transfected into HEK293F protein-expressing

cells in the same manner as described in Example 1. Next,

purification of the intact IgG-format cytosol-penetrating

antibody was performed in the same manner as described in

Example 1.

FIG. 33b shows a bar graph showing the results of

observing the cytosolic localization of an intact IgG-format cytosol-penetrating antibody, in which an improved endosomal escape motif has been introduced into a heavy-chain variable region thereof and an improved endosomal escape motif has been introduced into a light-chain variable region of a conventional therapeutic antibody fused with an EpCAM targeting peptide, by confocal microscopy using calcein and quantifying the calcein fluorescence of the confocal micrographs.

Specifically, human colorectal cancer HCT116 cells

having a K-RAS G13D mutation were prepared in the same manner

as described in Example 2. The cells were incubated with 0.1,

0.25 and 0.5 pM of each of CT-ep4l and CT01-ep4l at 370C for

6 hours. After 4 hours, each well containing PBS or the

antibody was treated with 150 pM calcein and incubated at

370C for 2 hours. In the same manner as described in Example

2, the cells were washed with PBS and weakly acidic solution,

and then fixed. The nucleus was blue-stained with

Hoechst33342 and observed with a confocal microscope. In the

cells treated with varying concentrations of CT01-ep41, the

intensity of green calcein fluorescence localized in the

cytosol was stronger than that in the cells treated with CT

ep41.

FIG. 33c shows a graph quantitatively comparing the

number of cells that have taken up trypan blue depending on

pH by an intact IgG-format cytosol-penetrating antibody in which an improved endosomal escape motif has been introduced into a heavy-chain variable region thereof and an improved endosomal escape motif has been introduced into a light-chain variable region of a conventional therapeutic antibody fused with an EpCAM-targeting peptide.

Specifically, Ramos cells were attached to plates in the

same manner as described in Example 5. Then, the cells were

incubated with each of 1 pM CT-ep41 and CT01-ep41 0.5 in 200

pl of each of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES pH

7.4) (for maintaining a cytosolic pH of 7.4) and pH 5.5

buffer (HBSS(Welgene), 50 mM MES pH 5.5)(for maintaining an

early endosomal pH of 5.5) at 370C for 2 hours. After careful

washing with PBS, 200 pl of a mixture of 190 pl of PBS and 10

pl of trypan blue was added to each well, and the cells were

observed with a microscope. The number of cells showing

trypan blue uptake was counted and expressed as percentage

relative to the total number of cells. A total of 400 or more

cells were counted, and the mean values are graphically shown.

As a result, CT01-ep41 showed a concentration-dependent

increase in trypan blue uptake compared to CT-ep41.

Thus, it was confirmed that when the endosomal escape

motif was introduced into each of the heavy-chain variable

region and the light-chain variable region, the endosomal

escape ability was improved compared to when the endosomal escape motif was present only in the light-chain variable region.

Example 39: Logic of Possibility of Imparting Improved

Endosomal Escape Motif to Heavy-Chain Variable Region of

Conventional Therapeutic Antibody

Similar to the logic that the endosomal escape motif was

imparted to the light-chain variable region of conventional

therapeutic antibodies, the use of the stable backbones of

commercially available therapeutic antibody can be expected

to increase the overall expression yield. In order to examine

whether the endosomal escape motif can operate as a single

motif, the endosomal escape motif was also imparted to the

heavy-chain variable region of conventional therapeutic

antibodies.

Mutants were constructed by substituting the CDR3 of

candidate heavy-chain variable regions with the CDR3 of the

cytosol-penetrating antibody.

Table 20 below shows the names and sequences of the

mutants constructed using a gene synthesis technique.

[Table 20]

Sequence SEQ ID NO: VariameRe ion 1 10 20 30 40 50 SEQ ID Humira-01 VH EV0LVESGGGLVQPGRSLRLSCAASFTFDDYAMHNVROAPGKGLEWVSAITWNSGHIDY 60 70 80 90 110 N0:54 ADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKGWYWMDLWGQGTLVTVSS 1 10 20 30 40 50 SEQ ID Herceptin-01 VH EVOLVESGGGLV0PGGSLRLSCAASGFNIKDTYIHIWVRQAPGKGLEWVARIYPTNGYTRY 60 70 80 90 110 NO:55 ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRGWYWMDLWGQGTLVTVSS 1 10 20 30 40 50 SEQ ID Avastin-01 VH EVOLVESGGGLV0PGGSLRLSCAASYTFTNYGMNWVRAPGKGLEWVGWINTYTGEPTYA 60 70 80 90 110 NO:56 ADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKGWYWMDLWGOGTLVTVSS

FIG. 34a is a schematic view showing a process of

constructing an intact IgG-format cytosol-penetrating

antibody in which an improved endosomal escape motif has been

introduced into the heavy-chain variable region of a

conventional therapeutic antibody.

In the same manner as described in Example 1, cloning of

the heavy-chain variable region was performed, and the

resulting heavy chain and the light chain comprising the

monoclonal antibody light-chain variable region introduced

with the improved endosomal escape motif were expressed

together in HEK293F cell lines and purified.

Example 40: Confirmation of Possibility of Imparting

Improved Endosomal Escape Motif to Heavy-Chain Variable

Region of Monoclonal Antibody

FIG. 34b is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by an

intact IgG-format cytosol-penetrating antibody in which an

improved endosomal escape motif has been introduced into the

heavy-chain variable region of a conventional therapeutic

antibody.

Specifically, pgsD-677 cells were prepared in the same

manner as described in Example 26. Then, in the same manner

as in described Example 5, the cells were incubated with 0.5

or 1 pM of each of CT-3, CT-3_WWW, CT01-AAA, CT01_WWW-AAA,

and CT01-3, and CT01_WWW-3_WWW in 200 pl of each of pH 7.4

buffer (HBSS(Welgene), 50 mM HEPES pH 7.4 (cytosolic pH)) and

pH 5.5 buffer (HBSS(Welgene), 50 mM MES pH 5.5 (early

endosomal pH)) at 370C for 2 hours. After careful washing

with PBS, 200 pl of a mixture of 190 pl of PBS and 10 pl of

trypan blue was added to each well, and the cells were

observed with a microscope. Next, after careful washing with

PBS, the cells were lysed by adding 50 pl of 1% SDS (sodium

dodecyl sulfate) to each well. The cells were transferred to

a 96-well plate, and the absorbance at 590 nm was measured.

All the mutants showed trypan blue uptake similar to that of

CT01-ep41.

Example 41: Construction of the Heavy-Chain Variable

Region and Light-Chain Variable Region Introduced with

Aspartic Acid for Improving Properties of Cytosol-Penetrating

Antibody

In order to improve the endosomal escape ability of the

cytosol-penetrating antibody, the endosomal escape motif was

introduced into the CRD3 of each of the heavy-chain variable

region and the light-chain variable region. Due to the

hydrophobic amino acids (tryptophan (W) and tyrosine (Y) of

the endosomal escape motif, the cytosol-penetrating antibody

becomes hydrophobic. To offset this hydrophobicity, mutants

were constructed by substituting an amino acid adjacent to

the endosomal escape motif with negatively charged aspartic

acid. The mutants were constructed with reference to studies

where aspartic acid was introduced into the backbone and CDR

regions of antibody variable regions to increase the overall

stability of the antibody and reduce protein aggregation

caused by the high hydrophobicity of the antibody (Perchiacca

et al., 2011; Dudgeon et al., 2012).

Since the 3 2 nd, 33rd and 5 8th amino acids of the heavy

chain variable region are adjacent to the endosomal escape

motif of each of the heavy-chain variable region and the

heavy-chain variable region, these amino acids were

substituted with aspartic acid. The resulting amino acids

were named CT11 VH (F32D, S33D) or CT12 VH (F32D, S33D, Y58D).

Since the 27bth, 50th and 51th amino acids of the light

chain variable region are adjacent to the endosomal escape motif of each of the heavy-chain variable region and the heavy-chain variable region, these amino acids were substituted with aspartic acid. The resulting amino acids were named hT4-60 VL (L27bD), hT4-61 VL (W50D), hT4-62 VL

(W50D, A51D) or hT4-63 VL (L27Bd, W50D, A51D).

Here, the heavy-chain variable region and light-chain

variable regions used as templates for the mutants are

antibody variable regions introduced with the endosomal

escape motif while showing high yields in animal cell

expression systems, and these regions were named CT01 VH and

hT4-59 VL, respectively.

Tables 21 and 22 below the heavy-chain variable region

and light-chain variable mutant sequences obtained by

introducing aspartic acid into the backbone and CDR regions

of the antibody variable region.

[Table 21]

Variable Region Sequence SEQ ID NO:

10 20 30 40 50 52a EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFSMSWVRQAPGKGLEWVSYISRTSHTTY SEQ ID CT10 VH 60 70 80 82a 90 100a 110 NQ:57 YADSVKGRFTISRDNSKNTLYLMNSLRAEDTAVYYCARGWYWMDLWGQGTLVTVSS 10 20 30 40 50 52a SEQ ID CT11 VH EVQLVESGGGLVQPGGSLRLSCAASGFTDDDFSMSWVRQAPGKGLEWVSYISRTSHTTY 60 70 80 82a 90 100a 110 NO:58 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGWYWMDLWGQGTLVTVSS 10 20 30 40 50 52a EVQLVESGGGLVPGGSLRLSCAASGFTDDDFSMSWVRQAPGKGLEWVSYISRTSHTTD SEQ ID CT12 VH 60 70 80 82a 90 100a 110 NQ:59 YADSVKGRFTISRDNSKNTLYLQMNSLRA[DTAVYYCARGWYWMDLWGQGTLVTVSS

[Table 22]

Sequence SEQ ID NO: VariablRegion 1 10 20 abcdef 30 40 50 DIGMTQSPSSLSASVGDRVTITCKSSOSLLNSRDGKNYLAWYQKPGKAPKLLIYW SEQ ID hT4-59 VL 60 70 80 90 100 NO:60 ASTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYFCQQYWYWMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 DIGMTOSPSSLSASVGDRVTITCKSSOSDLNSRDGKNYLAWY0QKPGKAPKLLIYW SEQ ID hT4-60 VL 60 70 80 90 100 NO:61 ASTRESGVPSRFSGSGSGTDFTLTISSLPEDFATYFGQQYWYWMYTFGOGTKVEIKR 1 10 20 abcdef 30 40 50 SEQ ID hT4-61 VL DIGMTOSPSSLSASVGDRVTITCKSSOSLLNSROGKNYLAWYOQKPGKAPKLLIYD 60 70 80 90 100 N0:62 ASTRESGVPSRFSGSGSGTDFTLTISSLPEDFATYFCQQYWYWMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 DIGMTOSPSSLSASVGDRVTITCKSSQSLLNSRDGKNYLAWYQQKPGKAPKLLIYD SEQ ID hT4-62 VL 60 70 80 90 100 N0:63 DSTRESGVPSRFSGSGSGTDFTLTISSLOPEDFATYFCQQYWYWMYTFGQGTKVEIKR 1 10 20 abcdef 30 40 50 DIGMTOSPSSLSASVGDRVTITCKSSQSDLNSRDGKNYLAWYQKPGKAPKLLIYD SEQ ID hT4-63 VL 60 70 80 90 100 NO:64 DSTRESGVPSRFSGSGSGTDFTLTISSLPEDFATYFCQQYWYWMYTFGQGTKVEIKR

In the same manner as described in Example 1, cloning of

each heavy-chain variable region was performed, and the

resulting heavy chain and the light chain comprising the

monoclonal antibody light-chain variable region introduced

with the improved endosomal escape motif were expressed

together in HEK293F cell lines and purified.

Example 42: Confirmation of Improvement in Endosomal

Escape Ability of Intact IgG-Format Cytosol-Penetrating

Antibody Comprising the Heavy-Chain Variable Region and/or

Light-Chain Variable Region Introduced with Aspartic Acid

FIG. 35 is a graph quantitatively comparing the number

of cells that have taken up trypan blue depending on pH by an

intact IgG-format cytosol-penetrating antibody comprising a

light-chain variable region and/or a heavy-chain variable

region introduced with aspartic acid.

Specifically, pgsD-677 cells were prepared in the same

manner as described in Example 26. Then, in the same manner

as in described Example 5, the cells were incubated with 1 pM

of each of CT10-ep59, CT11-ep59, CT12-ep59, CT10-ep60, CT10

ep61, CT10-ep62, CT10-ep63, and CT12-ep63 in 200 pl of each

of pH 7.4 buffer (HBSS(Welgene), 50 mM HEPES pH 7.4

(cytosolic pH)) and pH 5.5 buffer (HBSS(Welgene), 50 mM MES

pH 5.5 (early endosomal pH)) at 370C for 2 hours. After

careful washing with PBS, 200 pl of a mixture of 190 pl of

PBS and 10 pl of trypan blue was added to each well, and the

cells were observed with a microscope. Next, after careful

washing with PBS, the cells were lysed by adding 50 pl of 1%

SDS (sodium dodecyl sulfate) to each well. The cells were

transferred to a 96-well plate, and the absorbance at 590 nm

was measured. All the mutants showed trypan blue uptake

similar to that of CT01-ep41 tested in the above Example.

This suggests that even when aspartic acid is introduced, the

endosomal escape ability is not reduced. Antibody stability

experiments for these antibodies will be carried out later.

Example 43: Analysis of Structure of Cytosol-Penetrating

Antibody

In order to identify the structures of the IgG-format

cytosol-penetrating antibodies, the CT-59 antibody showing a

very high production yield was used among the cytosol

penetrating antibodies having endosomal escape ability at

endosomal acidic pH conditions. This antibody is an IgG

format cytosol-penetrating antibody comprising hTO VH and

hT4-59 VL as the heavy-chain variable region and the light

chain variable region, respectively.

To identify the three-dimensional structure, IgG-format

CT-59 produced using HEK293 cells was treated with papain,

and then high-purity Fab was purified by protein A column and

size exclusion chromatography. Next, a crystal for structural

identification was formed using an Mosquito-LCP system under

screening buffer index G1 conditions (0.2 M NaCl, 0.1 M Tris,

pH 8.5, 25 % (w/v) PEG3350). When the cytosol-penetrating

antibody was mixed with the screening buffer, the final pH

was 8.1.

FIG. 36a shows the results of observing a crystal of CT

59 Fab, formed under Index G1 conditions, by RI1000 (Rock

ImagerlOO; automatic protein crystal image analysis system).

X-ray diffraction data were collected at the 5C beamline

(Pohang Accelerator Laboratory(PAL)), and indexing and

scaling were performed using the HKL2000 package (HKL

Research Inc., USA), and then the Initial electron density

map of CT-59 Fab was obtained by a molecular replacement (MR)

method. The three-dimensional structure data of a protein

having a similar to that of CT-59 is required to use the MR

method, and a structure model obtained through the FFAS site

(http://ffas.sanfordburnham.org/ffas-cgi/cgi/ffas.pl) was

used as a model. Initial phase information of CT-59 Fab was

obtained using CCP4. Based on the obtained initial phase

information, a model building operation was performed using

COOT (Crystallographic Object-Oriented Toolkit,

http://www.biop.ox.ac.uk/coot/)), and refinement and

validation operations were performed using Refmac5

(http://www.ccp4.ac.uk/html/refmac5.html) and PHENIX (Python

based Hierarchical ENvironment for Integrated Xtallography,

http:// www.phenix-online.org/) software (see FIG. 36b).

As a result, at a final pH of pH 8.1, a three

dimensional structure with a high resolution of 1.8A was

observed. It was found that the distance between the 1st

aspartic acid (D) of the light-chain variable region of CT-59

and the side chain of the 9 5 th methionine (M) of the light

chain variable region was 6.87 A.

Although the present disclosure has been described in

detail with reference to the specific features, it will be

apparent to those skilled in the art that this description is

only for a preferred embodiment and does not limit the scope of the present disclosure. Thus, the substantial scope of the present disclosure will be defined by the appended claims and equivalents thereof.

Claims (19)

1. [Claims]

   [Claim 1]

   A cytosol-penetrating antibody or antigen-binding

   fragment thereof comprising a light-chain variable region

   and/or heavy-chain variable region that comprises an

   endosomal escape motif in its CDR3, wherein the endosomal

   escape motif comprises a sequence represented by the

   following formula:

   X1-X2-X3-Z1

   wherein each of Xl, X2 and X3 is selected from the group

   consisting of tryptophan (W), tyrosine (Y), histidine (H) and

   phenylalanine (F), wherein the endosomal escape motif

   comprise one or more tryptophan (W);

   Z1 is selected from the group consisting of methionine

   (M), isoleucine (I), leucine (L), histidine (H), aspartic

   acid (D), and glutamic acid (E);

   the 1st amino acid of the light-chain variable region

   and/or heavy-chain variable region is aspartic acid (D) or

   glutamic acid (E);

   the light-chain variable region and/or heavy-chain

   variable region comprising Z1 induces a change in properties

   of the antibody under endosomal acidic pH conditions; and

   the antibody exhibits an ability to escape from

   endosomes into the cytosol through the change in properties

   of the antibody.
2. [Claim 2]

   The cytosol-penetrating antibody or antigen-binding

   fragment thereof of claim 1, wherein the 1st amino acid of

   the light-chain variable region and/or heavy-chain variable

   region interacts with the Z1 under endosomal acidic pH

   conditions to induce a change in properties of the cytosol

   penetrating antibody.
3. [Claim 3]

   The cytosol-penetrating antibody or antigen-binding

   fragment thereof of claim 1 or 2, wherein the endosomal

   escape motif X1-X2-X3 of the light-chain variable region

   and/or heavy-chain variable region comprises one or more

   tryptophans.
4. [Claim 4]

   The cytosol-penetrating antibody or antigen-binding

   fragment thereof of any one of claims 1 to 3, wherein the

   endosomal escape motif X1-X2-X3 of the light-chain variable

   region and/or heavy-chain variable region comprises a sequence

   selected from the group consisting of W-W-W, W-W-H, W-Y-W, Y

   W-W, W-Y-H, and Y-W-H (where W is tryptophan, Y is tyrosine, H

   is histidine)
5. [Claim 5]

   The cytosol-penetrating antibody or antigen-binding

   fragment thereof of any one of claims 1 to 4, wherein the

   light-chain variable region and/or heavy-chain variable region further comprises an amino acid sequence represented by (al-...-an, where n is an integer ranging from 1 to 10) between X3 and Z1.
6. [Claim 61

   The cytosol-penetrating antibody or antigen-binding

   fragment thereof of any one of claims 1 to 5, wherein the

   sequence further comprises Z2 linked to Xl, and thus is

   represented by the following formula:

   Z2-X1-X2-X3-Z1,

   wherein Z2 is selected from the group consisting of

   glutamine (Q), leucine (L) histidine (H).
7. [Claim 7]

   The cytosol-penetrating antibody or antigen-binding

   fragment thereof of any one of claims 1 to 6, wherein the 1st

   amino acid of the light-chain variable region and/or heavy

   chain variable region interacts with Z1 and/or Z2 under

   endosomal acidic pH conditions to induce a change in

   properties of the cytosol-penetrating antibody.
8. [Claim 8]

   The cytosol-penetrating antibody or antigen-binding

   fragment thereof of any one of claims 1 to 7, wherein the

   light-chain variable region and/or heavy-chain variable region

   further comprises an amino acid sequence represented by (bl

   ...- bn, where n is an integer ranging from 1 to 10) between Xl

   and Z2.
9. [Claim 9]

   The cytosol-penetrating antibody or antigen-binding

   fragment thereof of any one of claims 1 to 8, wherein the

   CDR3 of the light-chain variable region comprises the

   sequence selected from the group consisting of SEQ ID NOS: 8

   to 12, 24 or 51.
10. [Claim 10]

    The cytosol-penetrating antibody or antigen-binding

    fragment thereof of any one of claims 1 to 9, wherein the

    CDR3 of the heavy-chain variable region comprises the

    sequence selected from the group consisting of SEQ ID NOS: 46

    to 49, and 53.
11. [Claim 11]

    The cytosol-penetrating antibody or antigen-binding

    fragment thereof of any one of claims 1 to 10, wherein the

    light-chain variable region comprise a sequence having a

    homolog of at least 80% to a light-chain variable region

    sequence selected from the group consisting of SEQ ID NOS: 1

    to 5, 13 to 23, 25 to 37, 50, and 60 to 64.
12. [Claim 12]

    The cytosol-penetrating antibody or antigen-binding

    fragment thereof of any one of claims 1 to 11, wherein the

    heavy-chain variable region comprise a sequence having a

    homolog of at least 80% to a heavy-chain variable region sequence selected from the group consisting of SEQ ID NOS: 39 to 42, 52, and 54 to 59.
13. [Claim 13]

    The cytosol-penetrating antibody or antigen-binding

    fragment thereof of any one of claims 1 to 12, wherein the

    antibody is an intact immunoglobulin G-format antibody.
14. [Claim 14]

    A nucleic acid encoding the antibody or antigen-binding

    fragment thereof of any one of claims 1 to 13.
15. [Claim 15]

    A vector comprising the nucleic acid of claim 14.
16. [Claim 16]

    A cell transformed with the vector of claim 15.
17. [Claim 17]

    A composition for delivering an active substance into

    cytosol, comprising the cytosol-penetrating antibody or

    antigen-binding fragment thereof of any one of claims 1 to 13.
18. [Claim 18]

    The composition of claim 17, wherein the active

    substance comprises one or more selected from the group

    consisting of peptides, proteins, toxins, antibodies,

    antibody fragments, RNAs, siRNAs, DNAs, small molecule drugs,

    nanoparticles, and liposomes.
19. [Claim 19]

    A method for producing the cytosol-penetrating antibody or

    antigen-binding fragment thereof of any one of claims 1 to 13,

    the method comprising a step of grafting the endosomal escape

    motif X1-X2-X3-Z1 (wherein X1-X2-X3 is selected from the

    group consisting of tryptophan (W), tyrosine (Y), histidine

    (H), and phenylalanine (F), wherein the endosomal escape

    motif comprise one or more tryptophan (W)) into the CDR3 of a

    light-chain and/or heavy-chain variable region.

    【Drawings】

    【Figure 1】

    【Figure 2a】

    1/34

    【Figure 2b】

    【Figure 2c】

    2/34

    【Figure 2d】

    【Figure 3a】

    3/34

    【Figure 3b】

    【Figure 3c】

    4/34

    【Figure 4】

    5/34

    【Figure 5】

    【Figure 6a】

    6/34

    【Figure 6b】

    【Figure 7a】

    7/34

    【Figure 7b】

    【Figure 8】

    8/34

    【Figure 9】

    【Figure 10】

    9/34

    【Figure 11】

    10/34

    【Figure 12】

    【Figure 13】

    11/34

    【Figure 14a】

    【Figure 14b】

    12/34

    【Figure 15a】

    【Figure 15b】

    【Figure 16】

    13/34

    【Figure 17a】

    【Figure 17b】

    【Figure 18】

    14/34

    【Figure 19】

    【Figure 20a】

    15/34

    【Figure 20b】

    【Figure 21a】

    16/34

    【Figure 21b】

    【Figure 21c】

    17/34

    【Figure 22a】

    【Figure 22b】

    18/34

    【Figure 22c】

    【Figure 23a】

    19/34

    【Figure 23b】

    【Figure 23c】

    20/34

    【Figure 24】

    【Figure 25a】

    21/34

    【Figure 25b】

    【Figure 26a】

    【Figure 26b】

    22/34

    【Figure 27】

    【Figure 28a】

    23/34

    【Figure 28b】

    【Figure 28c】

    24/34

    【Figure 29a】

    【Figure 29b】

    25/34

    【Figure 29c】

    【Figure 30a】

    【Figure 30b】

    26/34

    【Figure 30c】

    【Figure 30d】

    27/34

    【Figure 31a】

    【Figure 31b】

    28/34

    【Figure 32a】

    29/34

    【Figure 32b】

    【Figure 33a】

    30/34

    【Figure 33b】

    【Figure 33c】

    31/34

    【Figure 34a】

    【Figure 34b】

    32/34

    【Figure 35】

    【Figure 36a】

    33/34

    【Figure 36b】

    34/34

    PCTKR2017005559-seql.txt <110> Orum Therapeutics <120> Cytosol-Penetrating Antibodies and Uses Thereof <130> DP-2017-0135-KR

    <150> KR 10-2016-0065365 <151> 2016-05-27

    <150> KR 10-2016-0065379 <151> 2016-05-27 <160> 78 <170> KoPatentIn 3.0

    <210> 1 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-WWH VL

    <400> 1 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Trp Trp His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 2 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-WYW VL

    <400> 2 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Page 1

    PCTKR2017005559-seql.txt Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 3 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-YWW VL

    <400> 3 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Trp Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 4 <211> 114 <212> PRT <213> Artificial Sequence

    Page 2

    PCTKR2017005559-seql.txt <220> <223> hT4-WYH VL

    <400> 4 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Trp Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 5 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-YWH VL

    <400> 5 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Tyr Trp His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    Page 3

    PCTKR2017005559-seql.txt

    <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR1

    <400> 6 Lys Ser Ser Gln Ser Leu Phe Asn Ser Arg Thr Arg Lys Asn Tyr Leu 1 5 10 15

    Ala

    <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2

    <400> 7 Trp Ala Ser Thr Arg Glu Ser 1 5

    <210> 8 <211> 9 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-WWH VL

    <400> 8 Gln Gln Tyr Trp Trp His Met Tyr Thr 1 5

    <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> hT4-WYW VL

    <400> 9 Gln Gln Tyr Trp Tyr Trp Met Tyr Thr 1 5

    <210> 10 <211> 9 Page 4

    PCTKR2017005559-seql.txt <212> PRT <213> Artificial Sequence

    <220> <223> hT4-YWW VL

    <400> 10 Gln Gln Tyr Tyr Trp Trp Met Tyr Thr 1 5

    <210> 11 <211> 9 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-WYH VL

    <400> 11 Gln Gln Tyr Trp Tyr His Met Tyr Thr 1 5

    <210> 12 <211> 9 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-YWH VL

    <400> 12 Gln Gln Tyr Tyr Trp His Met Tyr Thr 1 5

    <210> 13 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-RYR VL

    <400> 13 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Page 5

    PCTKR2017005559-seql.txt 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Arg Tyr Arg Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 14 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-IYI VL

    <400> 14 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Ile Tyr Ile Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 15 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-GYG VL

    <400> 15 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Page 6

    PCTKR2017005559-seql.txt Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Gly Tyr Gly Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 16 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 VL

    <400> 16 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95

    Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 17 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 D1E-M95L VL

    Page 7

    PCTKR2017005559-seql.txt <400> 17 Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Trp Tyr Trp Leu Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 18 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-3 Y91H VL

    <400> 18 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    His Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 19 <211> 114 Page 8

    PCTKR2017005559-seql.txt <212> PRT <213> Artificial Sequence

    <220> <223> hT4-3 Y91D VL

    <400> 19 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Asp Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 20 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-3 L2E Q90L VL

    <400> 20 Asp Glu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Leu 85 90 95

    Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Page 9

    PCTKR2017005559-seql.txt Lys Arg

    <210> 21 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-3 L2E T97I VL

    <400> 21 Asp Glu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Trp Tyr Trp Met Tyr Ile Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 22 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 Q90H M95A VL

    <400> 22 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Page 10

    PCTKR2017005559-seql.txt 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His 85 90 95 Tyr Trp Tyr Trp Ala Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 23 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-3 Q90H VL

    <400> 23 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His 85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> hT4 3 Q90H VL

    <400> 24 Gln His Tyr Trp Tyr Trp Met Tyr Thr 1 5

    <210> 25 <211> 113 Page 11

    PCTKR2017005559-seql.txt <212> PRT <213> Artificial Sequence

    <220> <223> hT4-3 L8-1 VL

    <400> 25 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95

    Tyr Trp Tyr Trp Met Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

    Arg

    <210> 26 <211> 113 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-3 L8-2 VL

    <400> 26 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95

    Trp Tyr Trp Met Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Page 12

    PCTKR2017005559-seql.txt Arg

    <210> 27 <211> 115 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-3 L10-1 VL

    <400> 27 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95

    Tyr Trp Tyr Trp Pro Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 100 105 110

    Ile Lys Arg 115

    <210> 28 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L10-2 VL

    <400> 28 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Page 13

    PCTKR2017005559-seql.txt 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Trp Tyr Trp Leu Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 100 105 110 Ile Lys Arg 115

    <210> 29 <211> 115 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-3 L10-3 VL

    <400> 29 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Trp Tyr Trp Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu 100 105 110 Ile Lys Arg 115

    <210> 30 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L11-1 VL

    <400> 30 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Page 14

    PCTKR2017005559-seql.txt Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Trp Tyr Trp Leu Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Val 100 105 110

    Glu Ile Lys Arg 115

    <210> 31 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L11-2 VL

    <400> 31 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Pro Trp Tyr Trp Pro Met Tyr Thr Phe Gly Gln Gly Thr Lys Val 100 105 110 Glu Ile Lys Arg 115

    <210> 32 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Necitumumab-WYW VL

    Page 15

    PCTKR2017005559-seql.txt <400> 32 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45

    Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80

    Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Trp Tyr Trp Met Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Ala Glu Ile Lys Arg 100 105

    <210> 33 <211> 108 <212> PRT <213> Artificial Sequence

    <220> <223> Panitumumab-WYW VL

    <400> 33 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30

    Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45

    Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80

    Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Trp Tyr Trp Met Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105

    <210> 34 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Lumretuzumab-WYW VL

    Page 16

    PCTKR2017005559-seql.txt <400> 34 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95

    Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110

    Lys Arg

    <210> 35 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Nimotuzumab-WYW VL

    <400> 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Gln Gln Thr Pro Gly Lys Ala 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60

    Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile 65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr 85 90 95 Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr 100 105 110

    Arg

    <210> 36 Page 17

    PCTKR2017005559-seql.txt <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Emibetuzumab-WYW VL

    <400> 36 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Val Ser Ser Ile 20 25 30

    Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Trp Tyr Trp Met 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105

    <210> 37 <211> 108 <212> PRT <213> Artificial Sequence

    <220> <223> Pertuzumab-WYW VL

    <400> 37 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30

    Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Trp Tyr Trp Met Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105

    Page 18

    PCTKR2017005559-seql.txt <210> 38 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> HT0 VH

    <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30

    Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val 50 55 60

    Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr 65 70 75 80

    Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    Ala Arg Gly Ala Tyr Lys Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110

    Gly Thr Thr Val Thr Val Ser Ser 115 120

    <210> 39 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> HT0-01 VH

    <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

    Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val 50 55 60

    Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    Page 19

    PCTKR2017005559-seql.txt Ala Arg Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Thr Val 100 105 110

    Thr Val Ser Ser 115

    <210> 40 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> HT0-02 VH

    <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30

    Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

    Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val 50 55 60

    Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    Ala Arg Gly Trp Tyr Trp Phe Asp Leu Trp Gly Gln Gly Thr Thr Val 100 105 110

    Thr Val Ser Ser 115

    <210> 41 <211> 117 <212> PRT <213> Artificial Sequence

    <220> <223> HT0-03 VH

    <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30

    Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

    Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val 50 55 60 Page 20

    PCTKR2017005559-seql.txt Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Tyr Trp Gly Phe Asp Leu Trp Gly Gln Gly Thr Thr 100 105 110

    Val Thr Val Ser Ser 115

    <210> 42 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> HT0-04 VH

    <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30

    Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

    Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val 50 55 60

    Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr 65 70 75 80

    Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    Ala Arg Tyr Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115

    <210> 43 <211> 5 <212> PRT <213> Artificial Sequence

    <220> <223> CDR1

    <400> 43 Ser Tyr Val Met His 1 5

    Page 21

    PCTKR2017005559-seql.txt <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2

    <400> 44 Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly

    <210> 45 <211> 11 <212> PRT <213> Artificial Sequence

    <220> <223> HT0 VH

    <400> 45 Gly Ala Arg Lys Arg Gly Tyr Ala Met Asp Tyr 1 5 10

    <210> 46 <211> 7 <212> PRT <213> Artificial Sequence

    <220> <223> HT0-01 VH

    <400> 46 Gly Trp Tyr Trp Met Asp Leu 1 5

    <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HT0-02 VH

    <400> 47 Gly Trp Tyr Trp Phe Asp Leu 1 5

    <210> 48 <211> 8 <212> PRT <213> Artificial Sequence Page 22

    PCTKR2017005559-seql.txt <220> <223> HT0-03 VH

    <400> 48 Gly Trp Tyr Trp Gly Phe Asp Leu 1 5

    <210> 49 <211> 7 <212> PRT <213> Artificial Sequence

    <220> <223> HT0-04 VH

    <400> 49 Tyr Trp Tyr Trp Met Asp Leu 1 5

    <210> 50 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-3 WWW VL

    <400> 50 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95 Tyr Trp Trp Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 51 <211> 9 <212> PRT <213> Artificial Sequence Page 23

    PCTKR2017005559-seql.txt <220> <223> hT4 3 WWW VL

    <400> 51 Gln Gln Tyr Trp Trp Trp Met Tyr Thr 1 5

    <210> 52 <211> 116 <212> PRT <213> Artificial Sequence

    <220> <223> HT0-01 WWW VH

    <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30

    Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

    Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr 65 70 75 80

    Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    Ala Arg Gly Trp Trp Trp Met Asp Leu Trp Gly Gln Gly Thr Thr Val 100 105 110

    Thr Val Ser Ser 115

    <210> 53 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HT0-01 WWW VH

    <400> 53 Gly Trp Trp Trp Met Asp Leu 1 5

    <210> 54 <211> 116 <212> PRT <213> Artificial Sequence Page 24

    PCTKR2017005559-seql.txt <220> <223> Humira-01 VH

    <400> 54 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

    Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115

    <210> 55 <211> 116 <212> PRT <213> Artificial Sequence

    <220> <223> Herceptin-01 VH

    <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30

    Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Page 25

    PCTKR2017005559-seql.txt 115

    <210> 56 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Avastin-01 VH

    <400> 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

    Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60

    Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80

    Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    Ala Lys Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val 100 105 110

    Thr Val Ser Ser 115

    <210> 57 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> CT10 VH

    <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30

    Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

    Ser Tyr Ile Ser Arg Thr Ser His Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80

    Page 26

    PCTKR2017005559-seql.txt Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    Ala Arg Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val 100 105 110

    Thr Val Ser Ser 115

    <210> 58 <211> 116 <212> PRT <213> Artificial Sequence

    <220> <223> CT11 VH

    <400> 58 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Asp Asp Asp Phe 20 25 30

    Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

    Ser Tyr Ile Ser Arg Thr Ser His Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80

    Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    Ala Arg Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val 100 105 110

    Thr Val Ser Ser 115

    <210> 59 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> CT12 VH

    <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Asp Asp Asp Phe 20 25 30

    Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Page 27

    PCTKR2017005559-seql.txt Ser Tyr Ile Ser Arg Thr Ser His Thr Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    Ala Arg Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115

    <210> 60 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-59 VL

    <400> 60 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30

    Arg Asp Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 61 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-60 VL

    <400> 61 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Page 28

    PCTKR2017005559-seql.txt 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Asp Leu Asn Ser 20 25 30 Arg Asp Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95

    Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 62 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-61 VL

    <400> 62 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Asp Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 63 <211> 114 <212> PRT <213> Artificial Sequence Page 29

    PCTKR2017005559-seql.txt <220> <223> hT4-62 VL

    <400> 63 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Asp Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Asp Asp Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 64 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-63 VL

    <400> 64 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Asp Leu Asn Ser 20 25 30

    Arg Asp Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Asp Asp Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Page 30

    PCTKR2017005559-seql.txt

    <210> 65 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4 VL

    <400> 65 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 66 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-D1A VL

    <400> 66 Ala Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Page 31

    PCTKR2017005559-seql.txt Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 67 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-M95A VL

    <400> 67 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Tyr Tyr His Ala Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 68 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-D1E VL

    <400> 68 Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Page 32

    PCTKR2017005559-seql.txt Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 69 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-M95L VL

    <400> 69 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Tyr His Leu Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 70 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-Y91A VL

    <400> 70 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Page 33

    PCTKR2017005559-seql.txt 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Ala Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 71 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-Y92A VL

    <400> 71 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Ala Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 72 <211> 114 <212> PRT <213> Artificial Sequence Page 34

    PCTKR2017005559-seql.txt <220> <223> hT4-Y93A VL

    <400> 72 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ala His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    <210> 73 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-H94A VL

    <400> 73 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Tyr Ala Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Page 35

    PCTKR2017005559-seql.txt

    <210> 74 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-AAA VL

    <400> 74 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Ala Ala Ala Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 75 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-Y96A VL

    <400> 75 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30

    Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Page 36

    PCTKR2017005559-seql.txt Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Tyr Tyr His Met Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 76 <211> 108 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-01 VL

    <400> 76 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Gly Leu Ser Ser Tyr 20 25 30

    Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45

    Tyr Trp Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80

    Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Tyr His Met Tyr 85 90 95

    Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105

    <210> 77 <211> 114 <212> PRT <213> Artificial Sequence

    <220> <223> hT4-02 VL

    <400> 77 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30

    Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Page 37

    PCTKR2017005559-seql.txt Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110

    Lys Arg

    <210> 78 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-03 VL

    <400> 78 Asp Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30

    Asp Asp Gly Asn Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45

    Ala Pro Lys Leu Leu Ile Tyr Trp Leu Ser Tyr Arg Ala Ser Gly Val 50 55 60

    Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

    Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95

    Tyr Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg

    Page 38